Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumoniae The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-α-l-Rhap-[4-P-1-Gro]-(1-3)-α-d-Glcp-[(6-P-1)-Gro]-(1-3)-β-l-Rhap-[2-OAc]-(1-4)-β-d-Glcp-(1-; 16A, -3)-β-d-Galf-[2-OAc (70%)]-(1-3)-α-l-Rhap-(1-2)-α-l-Rhap-(1-3)-α-d-Galp-[(6-P-1)-Gro]-(1-3)-β-d-Galp-(1-4)-β-d-Glcp-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published cps gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of α-l-Rhap-(1-3)-α-d-Glcp and α-l-Rhap-(1-2)-α-l-Rhap, respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with factor antiserum 11c.IMPORTANCE The vaccine pressure against Streptococcus pneumoniae could result in a change of prevalence in carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve successful vaccination of the population, and similarly, it is important to increase the knowledge of even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen, and antigenic properties of CPS are based on the structure. Consequently, a better understanding of the structure, biosynthesis, and serology of the capsular polysaccharides can be of great importance toward developing future diagnostic tools and vaccines.

Streptococcus pneumoniae, capsular polysaccharides, NMR spectroscopy, Structural characterization, serotypes 16F and 16A

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,3,4,0	x?Gro	67.04	71.26	62.70
4,3,3,4	P
4,3,3	aLRhap	100.69	70.56	80.99	75.72	68.33	17.34
4,3,6,0	x?Gro	67.04	71.26	62.70
4,3,6	P
4,3	aDGlcp	95.18	71.91	79.94	67.98	71.15	64.70
4,2	Ac	174.07	20.74
4	bLRhap	99.63	68.92	75.25	71.15	72.67	17.22
	bDGlcp	104.56	73.61	75.48	77.68	74.66	61.13


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,3,4,0	x?Gro	3.855-3.921	3.874	3.588-3.666
4,3,3,4	P
4,3,3	aLRhap	5.107	4.268	3.964	4.110	4.128	1.286
4,3,6,0	x?Gro	3.874	3.874	3.588-3.666
4,3,6	P
4,3	aDGlcp	5.000	3.657	3.788	3.543	3.985	4.058
4,2	Ac	-	2.154
4	bLRhap	5.037	5.634	3.866	3.504	3.506	1.350
	bDGlcp	4.536	3.373	3.580	3.654	3.447	3.755-3.860


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,3,4,0	x?Gro	67.04/3.855-3.921	71.26/3.874	62.70/3.588-3.666
4,3,3,4	P
4,3,3	aLRhap	100.69/5.107	70.56/4.268	80.99/3.964	75.72/4.110	68.33/4.128	17.34/1.286
4,3,6,0	x?Gro	67.04/3.874	71.26/3.874	62.70/3.588-3.666
4,3,6	P
4,3	aDGlcp	95.18/5.000	71.91/3.657	79.94/3.788	67.98/3.543	71.15/3.985	64.70/4.058
4,2	Ac		20.74/2.154
4	bLRhap	99.63/5.037	68.92/5.634	75.25/3.866	71.15/3.504	72.67/3.506	17.22/1.350
	bDGlcp	104.56/4.536	73.61/3.373	75.48/3.580	77.68/3.654	74.66/3.447	61.13/3.755-3.860

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](COP(=O)(O)OCC(O)CO)O[C@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@H](O[C@@H]4[C@@H](CO)O[C@@H](*)[C@H](O)[C@H]4O)[C@@H]3OC(C)=O)[C@@H]2O)O[C@@H](C)[C@@H]1OP(=O)(O)OCC(O)CO

966.719 g/mol (C₃₂H₅₆R₂O₂₉P₂, R = next and previous repeats, not counted in mol weight)