In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

O-antigen, NMR spectroscopy, O-polysaccharide, structure elucidation, lipopolysaccharide (LPS), MALDI-TOF mass spectrometry, Aeromonas, bacillosamine, Fish pathogen, D-QuiN4N

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3,2	Ac	175.5	23.7
3,3,3	aDGalpN	98.2	50.6	68.8	76.3	71.4	61.3
3,3,2	Ac	176.4	23.7
3,3,4	lS3HOBut	175.0	46.2	66.0	23.7
3,3	bDQuipN4N	103.3	56.2	75.9	58.0	72.3	17.7
3	bDGalp	106.1	71.2	82.8	69.6	75.9	62.3
2	Ac	176.4	23.7
	bDGalpN	103.3	52.8	80.8	69.5	75.9	62.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3,2	Ac	-	2.05
3,3,3	aDGalpN	5.16	4.12	3.89	4.21	3.87	3.73-3.87
3,3,2	Ac	-	2.03
3,3,4	lS3HOBut	-	2.35	4.20	1.25
3,3	bDQuipN4N	4.74	3.87	3.87	3.87	3.54	1.21
3	bDGalp	4.46	3.61	3.70	4.10	3.65	3.76-3.81
2	Ac	-	2.07
	bDGalpN	4.74	4.05	3.93	4.17	3.71	3.76-3.81


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3,2	Ac		23.7/2.05
3,3,3	aDGalpN	98.2/5.16	50.6/4.12	68.8/3.89	76.3/4.21	71.4/3.87	61.3/3.73-3.87
3,3,2	Ac		23.7/2.03
3,3,4	lS3HOBut		46.2/2.35	66.0/4.20	23.7/1.25
3,3	bDQuipN4N	103.3/4.74	56.2/3.87	75.9/3.87	58.0/3.87	72.3/3.54	17.7/1.21
3	bDGalp	106.1/4.46	71.2/3.61	82.8/3.70	69.6/4.10	75.9/3.65	62.3/3.76-3.81
2	Ac		23.7/2.07
	bDGalpN	103.3/4.74	52.8/4.05	80.8/3.93	69.5/4.17	75.9/3.71	62.3/3.76-3.81

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@@H](NC(C)=O)[C@H](O[C@H]3[C@@H](O)[C@@H](CO)O[C@@H](O[C@@H]4[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@@H]4O)[C@@H]3O)O[C@H](C)[C@H]2NC(=O)C[C@H](C)O)[C@H](NC(C)=O)[C@H]1O

840.83 g/mol (C₃₄H₅₆R₂N₄O₂₀, R = next and previous repeats, not counted in mol weight)