Shigella flexneri is the major human pathogen causing shigellosis. O-antigens of all S. flexneri serotypes (except for serotype 6) share the →2)-α-l-Rhap(III)-(1 → 2)-α-l-Rhap(II)-(1 → 3)-α-l-Rhap(I)-(1 → 3)-β-d-GlcpNAc-(1→ basic O-unit, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation at various positions. Recently, in serotype X and 4a variants called Xv and 4av, respectively, O-antigen modification with phosphoethanolamine (PEtN) has been identified, which is encoded by a plasmid-borne gene (lpt-O) for a PEtN-transferase and confers the monoclonal antibody IV-1(MASF IV-1) determinant to the bacteria. In this study, we elucidated the O-antigen structure of serotype Yv, another MASF IV-1-positive novel variant of S. flexneri. The serotype Yv O-antigen has the same basic carbohydrate backbone structure as that of the "classical" serotype Y, but differs in the presence of PEtN at position 3 of Rha(III) (major) or both Rha(II) and Rha(III) (minor). This pattern is similar to that of serotype 4av, but different from the pattern of serotype Xv, which is characterized by major PEtN modification on Rha(II). In serotype Yv, mono- and bisphosphorylated O-units generate a block-copolymeric structure, the former being partially O-acetylated at position 6 of GlcNAc and the latter lacking O-acetylation. Functional analysis revealed a correlation between the serotype-specific PEtN modification pattern and the lpt-O variation in different serotypes: lpt-O(RII) in serotype Xv is better tuned for phosphorylation of Rha(II) and lpt-O(RIII) in serotypes Yv and 4av for phosphorylation of Rha(III). These data enhance our knowledge of S. flexneri serotype conversion mechanisms and help to understand the biosynthesis process of the new O-antigen variants.

O-antigen, Shigella flexneri, polysaccharide structure, serotype conversion, phosphoethanolamine modification, phosphoethanolamine transferase gene

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,2,3	aLRhap	102.5	71.9	71.4	73.2	70.1	17.7
2,2,2	Ac	175.7	23.5
2,2	bDGlcpN	103.4	56.6	82.2	69.7	76.9	61.8
2,3,0	xXEtN	63.1	41.3
2,3	P
2	aLRhap	99.8	78.9	76.3	72.5	70.3	17.8
	x?Gro-al	90.3	81.5	60.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,2,3	aLRhap	4.89	3.83	3.75	3.44	3.98	1.25
2,2,2	Ac	-	2.11
2,2	bDGlcpN	4.71	3.88	3.66	3.54	3.49	3.76-3.91
2,3,0	xXEtN	4.17	3.28
2,3	P
2	aLRhap	5.13	4.29	4.30	3.42	3.94	1.28
	x?Gro-al	5.10	3.65	3.74-3.86


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,2,3	aLRhap	102.5/4.89	71.9/3.83	71.4/3.75	73.2/3.44	70.1/3.98	17.7/1.25
2,2,2	Ac		23.5/2.11
2,2	bDGlcpN	103.4/4.71	56.6/3.88	82.2/3.66	69.7/3.54	76.9/3.49	61.8/3.76-3.91
2,3,0	xXEtN	63.1/4.17	41.3/3.28
2,3	P
2	aLRhap	99.8/5.13	78.9/4.29	76.3/4.30	72.5/3.42	70.3/3.94	17.8/1.28
	x?Gro-al	90.3/5.10	81.5/3.65	60.8/3.74-3.86

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@H]2[C@H](OC(C=O)CO)O[C@@H](C)[C@H](O)[C@H]2OP(=O)(O)OCCN)O[C@H](CO)[C@@H](O)[C@@H]1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O

708.604 g/mol (C₂₅H₄₅N₂O₁₉P)