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
4,3,3,2,3	bDGalf	109.79	80.54	85.20	82.46	70.73	63.34
4,3,3,2	aLRhap	102.39	70.57	78.13	71.54	69.77	17.21
4,3,3	aLRhap	101.11	78.61	70.73	72.66	69.93	17.21
4,3,6,0	x?Gro	62.54	71.21	66.88
4,3,6	P
4,3	aDGalp	96.29	68.32	76.84	68.96	70.09	64.46
4	bDGalp	103.36	70.09	78.13	65.59	75.55	61.41
	bDGlcp	102.39	73.14	74.75	78.93	75.39	60.43


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,3,2,3	bDGalf	5.230	4.342	4.264	4.178	3.943	3.656-3.708
4,3,3,2	aLRhap	4.941	4.170	3.857	3.513	3.752	1.252
4,3,3	aLRhap	5.191	4.057	3.943	3.493	3.802	1.287
4,3,6,0	x?Gro	3.583-3.650	3.877	3.842-3.896
4,3,6	P
4,3	aDGalp	5.124	3.967	3.998	4.111	4.334	3.951
4	bDGalp	4.526	3.642	3.767	4.147	3.712	3.744-3.775
	bDGlcp	4.639	3.329	3.665	3.669	3.611	3.963


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,3,2,3	bDGalf	109.79/5.230	80.54/4.342	85.20/4.264	82.46/4.178	70.73/3.943	63.34/3.656-3.708
4,3,3,2	aLRhap	102.39/4.941	70.57/4.170	78.13/3.857	71.54/3.513	69.77/3.752	17.21/1.252
4,3,3	aLRhap	101.11/5.191	78.61/4.057	70.73/3.943	72.66/3.493	69.93/3.802	17.21/1.287
4,3,6,0	x?Gro	62.54/3.583-3.650	71.21/3.877	66.88/3.842-3.896
4,3,6	P
4,3	aDGalp	96.29/5.124	68.32/3.967	76.84/3.998	68.96/4.111	70.09/4.334	64.46/3.951
4	bDGalp	103.36/4.526	70.09/3.642	78.13/3.767	65.59/4.147	75.55/3.712	61.41/3.744-3.775
	bDGlcp	102.39/4.639	73.14/3.329	74.75/3.665	78.93/3.669	75.39/3.611	60.43/3.963

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H]([C@H](O)CO)O[C@@H](O[C@@H]2[C@@H](O)[C@H](C)O[C@@H](O[C@H]3[C@H](O[C@H]4[C@@H](O)[C@@H](COP(=O)(O)OCC(O)CO)O[C@H](O[C@H]5[C@@H](O)[C@@H](CO)O[C@@H](O[C@@H]6[C@@H](CO)O[C@@H](*)[C@H](O)[C@H]6O)[C@@H]5O)[C@@H]4O)O[C@@H](C)[C@H](O)[C@H]3O)[C@@H]2O)[C@@H]1O

1094.906 g/mol (C₃₉H₆₇R₂O₃₃P, R = next and previous repeats, not counted in mol weight)