Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes

Abstract

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aalpha and 11Abeta. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11Aalpha was determined to be identical to 11A. 11Abeta is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11Aalpha and 11Abeta subtypes.

FIGURE 1.

Overlay of the ¹H-¹³C HSQC spectra of native (red) and de-O-acetylated (black) S. pneumoniae 11A PS. Peaks marked with an asterisk are peaks that show connectivities in the HMBC spectra and do not shift markedly between the two samples. Peaks marked with the pound sign are involved in connectivities as indicated in the HMBC spectra. The black arrows indicate the four anomeric resonances observed for the core polysaccharide.

FIGURE 2.

¹H-¹³C HMBC spectrum of de-O-acetylated S. pneumoniae 11A PS. Cross-peaks showing connectivities are indicated in the figure. Nomenclature is indicated in Table 1.

FIGURE 3.

¹H-³¹P HMBC of different S. pneumoniae PS. A, ST11A native (red) and de-O-acetylated (black). B, ST11A native (red) and ST11Aβ native (green). C, de-O-acetylated ST11A (red) and ST11Aβ (green). D, ST11Aβ native (green) and de-O-acetylated (black).

FIGURE 4.

Revised structure of de-O-acetylated S. pneumoniae 11A PS.

FIGURE 5.

Overlay of the ¹H-¹³C HMBC spectra of native (red) and de-O-acetylated (black) 11A PS. Spectra were acquired with identical parameters, 140 Hz ¹J_CH, and 8 Hz multiple bond coupling (125-ms delay).

FIGURE 6.

Regions of the ¹H-¹³C HMBC spectrum of native S. pneumoniae 11A PS showing connectivities for three acetate peaks. A, connectivities from the acetate methyl to carbon chemical shift of substituted residues. B, connectivities from the substituted residues to the carbonyls of the acetate. There is no connection for the carbonyl from the peak at 5.59 ppm.

FIGURE 7.

Revised structure of native S. pneumoniae 11A PS and 11Aα PS.

FIGURE 8.

Overlay of the ¹H-¹³C HSQC of ST11A and ST11Aβ. The box indicates the H-1 peaks of the acetylated glycerol, which are missing in ST11Aβ. See text for further details.

FIGURE 9.

Structure of ST11Aβ.