Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D- N -acetylglucosamine

Abstract

For many bacteria, including those important in pathogenesis, expression of a surface-localized capsular polysaccharide (CPS) can be critical for survival in host environments. In Gram-positive bacteria, CPS linkage is to either the cytoplasmic membrane or the cell wall. Despite the frequent occurrence and essentiality of these polymers, the exact nature of the cell wall linkage has not been described in any bacterial species. Using the Streptococcus pneumoniae serotype 2 CPS, which is synthesized by the widespread Wzy mechanism, we found that linkage occurs via the reducing end glucose of CPS and the β-D-N-acetylglucosamine (GlcNAc) residues of peptidoglycan (PG). Hydrofluoric acid resistance, 31 P-NMR, and 32 P labeling demonstrated the lack of phosphodiester bonds, which typically occur in PG-polysaccharide linkages. Component sugar analysis of purified CPS-PG identified only CPS and PG sugars in the appropriate ratios, suggesting the absence of an oligosaccharide linker. Time of flight mass spectrometry confirmed a direct glycosidic linkage between CPS and PG and showed that a single CPS repeat unit can be transferred to PG. The linkage was acetolysis susceptible, indicative of a 1,6 glycosidic bond between CPS and the GlcNAc C-6. The acetylation state of GlcNAc did not affect linkage. A direct glycosidic linkage to PG was also demonstrated for serotypes 8 and 31, whose reducing end sugars are glucose and galactose, respectively. These results provide the most detailed descriptions of CPS-PG linkages for any microorganism. Identification of the linkage is a first step toward identifying the linking enzyme and potential inhibitors of its activity.capsule | Wzy | Gram positive | pneumococcus | glycobiology

Progress toward a group B streptococcal vaccine

“…High molecular weight CPS is covalently linked to GlcNAc C-6 of peptidoglycan, but the genetic and enzymatic mechanisms involved in polysaccharide-PGN ligation remain unclear. 61…”

Quality Improvement of Capsular Polysaccharide in Streptococcus pneumoniae by Purification Process Optimization

“…Because CWPS is known to cause an inflammatory response (Tuomanen et al, 1987) and antibodies against CWPS are known to be not protective despite its high immunogenicity (Nielsen et al, 1993), CWPS must be separated from CPS for pneumococcal vaccine development. However, it is difficult to remove CWPS from CPS because the molecules are covalently bound through peptidoglycan (Sørensen et al, 1990;Larson and Yother, 2017) except for serotypes 3 and 37. In this study, we developed an improved process for CPS purification from the fermentation broth of S. pneumoniae that reduces CWPS as well as other contaminants such as nucleic acids and proteins.…”

Membrane linkage of aStreptococcus pneumoniaeWzy capsular polysaccharide occurs through an acylglycerol

“…In S. pneumoniae, the completed CPS polymer exhibits a broad range in size, from only a few repeat units to as many as 2000 repeat units (16,20,21). Similar, if not identical, size distributions of polymer are seen on membrane and peptidoglycan fractions for multiple serotypes (16), suggesting that transfer may be random and lacking in a requirement for a termination step that requires a long-chain polymer.…”

“…Unlike the linkages of other polysaccharides to peptidoglycan, where a phosphodiester bond is involved (22)(23)(24)(25)(26)(27)(28), linkage of the serotype 2 CPS to peptidoglycan occurs via a direct 1,6 glycosidic linkage between the reducing end Glc of CPS and the β-D-N-acetylglucosamine (GlcNAc) of peptidoglycan ( Fig. 1) (20). The serotype 8 and 31 capsules, whose reducing-end sugars are Glc and galactose respectively, are also attached to peptidoglycan through direct glycosidic linkages (20).…”

“…1) (20). The serotype 8 and 31 capsules, whose reducing-end sugars are Glc and galactose respectively, are also attached to peptidoglycan through direct glycosidic linkages (20). There have been no published reports suggestive of CPS transfer from Und-P to an alternate membrane acceptor, and it is generally assumed that capsule detected on the membrane is still in the active process of synthesis prior to transfer to peptidoglycan or release from the cell (the latter of which also lacks a description of its mechanism).…”