Vibrio anguillarum is a Gram-negative bacterium associated with vibriosis in Atlantic cod (Gadus morhua L.). Although farmed cod in Norway is routinely vaccinated against the infection, outbreaks of V. anguillarum-associated vibriosis still occur. Here, we describe the structural characterization of the LPS O-chain polysaccharide (O-PS) from atypical isolates of V. anguillarum strain 1282 and show that it is distinct from that previously established for V. anguillarum serotype O2. The structure of the purified O-PS was shown by 1D/2D NMR ((1)H, (13)C) spectroscopy and CE-MS studies to be a high-molecular mass linear polymer of tetrasaccharide repeating units, composed of 2-acetamido-3-(N-formyl-l-alanyl)amido-2,3-dideoxy-d-glucuronamide [GlcNAc3N(Fo-L-Ala)AN], 2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid (ManNAc3NAmA), 3-acetamido-3-dideoxy-d-quinovose (Qui3NAc), and 2,4-diacetamido-2,4-dideoxy-d-fucose (FucNAc4NAc). NMR analysis of the partial hydrolysis-derived oligosaccharides confirmed the presence of an O-acetyl group at position O-4 of GlcNAc3N(Fo-l-Ala)AN and established that the above-mentioned structure represents the biological repeating unit of the O-PS. In addition, it was demonstrated that some of 2,3-diamino-2,3-dideoxy-glucuronamide in the O-PS was present in the form of 2,3-diamino-2,3-dideoxy-glucose.

structure, O-antigen, serotyping, Vibrio anguillarum, CE-MS, vibriosis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,3	xLAla?	178.2	51.3	18.1
4,4,2	Ac	176.2-177.7	22.8-23.5
4,4,6	NH2
4,4	bDGlcpN3NA	103.2	54.4	55.3	70.6	77.2	173.9
4,2	Ac	176.2-177.7	22.8-23.5
4,3	Am	167.6	20.0
4	bDManpN3NA	101.2	51.0	55.9	76.0	77.2	173.2
3	Ac	176.2-177.7	22.8-23.5
	xDQui3N-ol	63.9	71.9	52.2	84.4	68.3	17.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,3	xLAla?	-	4.21	1.32
4,4,2	Ac	-	1.98-2.09
4,4,6	NH2
4,4	bDGlcpN3NA	4.65	3.76	4.02	3.68	4.07	-
4,2	Ac	-	1.98-2.09
4,3	Am	?	2.17
4	bDManpN3NA	5.05	4.67	4.07	4.04	4.01	-
3	Ac	-	1.98-2.09
	xDQui3N-ol	3.54	3.83	4.04	3.88	3.97	1.15


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,3	xLAla?		51.3/4.21	18.1/1.32
4,4,2	Ac		22.8-23.5/1.98-2.09
4,4,6	NH2
4,4	bDGlcpN3NA	103.2/4.65	54.4/3.76	55.3/4.02	70.6/3.68	77.2/4.07	
4,2	Ac		22.8-23.5/1.98-2.09
4,3	Am	167.6/?	20.0/2.17
4	bDManpN3NA	101.2/5.05	51.0/4.67	55.9/4.07	76.0/4.04	77.2/4.01	
3	Ac		22.8-23.5/1.98-2.09
	xDQui3N-ol	63.9/3.54	71.9/3.83	52.2/4.04	84.4/3.88	68.3/3.97	17.4/1.15

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=N)N[C@H]1[C@H](O[C@@H]2O[C@H](C(N)=O)[C@@H](O)[C@H](NC(=O)[C@H](C)N)[C@H]2NC(C)=O)[C@@H](C(=O)O)O[C@@H](O[C@H]([C@@H](C)O)[C@H](NC(C)=O)[C@@H](O)CO)[C@H]1NC(C)=O

750.76 g/mol (C₂₉H₅₀N₈O₁₅)