Salmonella paratyphi A is increasingly recognized as a common cause of enteric fever cases and there are no licensed vaccines against this infection. Antibodies directed against the O-polysaccharide of the lipopolysaccharide of Salmonella are protective and conjugation of the O-polysaccharide to a carrier protein represents a promising strategy for vaccine development. O-Acetylation of S. paratyphi A O-polysaccharide is considered important for the immunogenicity of S. paratyphi A conjugate vaccines. Here, as part of a programme to produce a bivalent conjugate vaccine against both S. typhi and S. paratyphi A diseases, we have fully elucidated the O-polysaccharide structure of S. paratyphi A by use of HPLC-SEC, HPAEC-PAD/CD, GLC, GLC-MS, 1D and 2D-NMR spectroscopy. In particular, chemical and NMR studies identified the presence of O-acetyl groups on C-2 and C-3 of rhamnose in the lipopolysaccharide repeating unit, at variance with previous reports of O-acetylation at a single position. Moreover HR-MAS NMR analysis performed directly on bacterial pellets from several strains of S. paratyphi A also showed O-acetylation on C-2 and C-3 of rhamnose, thus this pattern is common and not an artefact from O-polysaccharide purification. Conjugation of the O-polysaccharide to the carrier protein had little impact on O-acetylation and therefore should not adversely affect the immunogenicity of the vaccine.

O-polysaccharide, O-acetylation, bacterial polysaccharide structure, Salmonella paratyphi A

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3	aXParp	100.2	67.6	35.1	70.4	69.9	17.2
3,4	aDManp	100.7	79.7	77.8	67.0	74.2	61.2
3	aLRhap	102.7	71.1	69.8	82.1	68.7	17.7-17.9
6	70%aDGlcp	99.6	72.0	73.8	70.1	72.9	61.3
	aDGalp	101.7	68.4	77.9	69.5	69.8	66.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3	aXParp	5.07	3.83	2.13	3.38	3.72	1.24
3,4	aDManp	5.18	4.02	4.05	4.05	3.96	3.83
3	aLRhap	5.06	4.08	3.97	3.60	3.96	1.34
6	70%aDGlcp	4.95	3.56	3.73	3.42	3.66	3.76-3.87
	aDGalp	5.24	3.95	3.96	4.16	4.23	3.69-3.79


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3	aXParp	100.2/5.07	67.6/3.83	35.1/2.13	70.4/3.38	69.9/3.72	17.2/1.24
3,4	aDManp	100.7/5.18	79.7/4.02	77.8/4.05	67.0/4.05	74.2/3.96	61.2/3.83
3	aLRhap	102.7/5.06	71.1/4.08	69.8/3.97	82.1/3.60	68.7/3.96	17.7-17.9/1.34
6	70%aDGlcp	99.6/4.95	72.0/3.56	73.8/3.73	70.1/3.42	72.9/3.66	61.3/3.76-3.87
	aDGalp	101.7/5.24	68.4/3.95	77.9/3.96	69.5/4.16	69.8/4.23	66.8/3.69-3.79

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](O[C@H]2O[C@H](C)[C@@H](O)C[C@H]2O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@H]1[C@H](C)O[C@@H](O[C@@H]2[C@@H](O)[C@@H](*)O[C@H](CO[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@@H]2O)[C@H](O)[C@@H]1O

762.708 g/mol (C₃₀H₅₀R₂O₂₂, R = next and previous repeats, not counted in mol weight)