Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→, O-PS2 →2)-α-l-Rhap4NAc-(1→. The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

O-antigen, NMR spectroscopy, O-polysaccharide, endotoxin, structure elucidation, lipopolysaccharide (LPS), MALDI-TOF mass spectrometry, Aeromonas hydrophila, L-perosamine, l-Rha4NAc

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,4	Ac	175.6-176.0	23.3-23.4
3,4	aLRhap4N	103.0	70.3	78.0	52.5	69.4	18.0
3,2	Ac	175.6-176.0	23.3-23.4
3	aDGalpN	95.0	50.9	69.2	77.2	72.7	62.2
4	Ac	175.6-176.0	23.3-23.4
	aLRhap4N	103.0	67.0	74.3	52.3	69.4	18.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,4	Ac	-	2.05-2.06
3,4	aLRhap4N	5.17	4.20	3.97	3.97	3.92	1.23
3,2	Ac	-	2.05-2.06
3	aDGalpN	5.04	4.26	3.97	4.12	3.90	3.74-3.81
4	Ac	-	2.05-2.06
	aLRhap4N	5.00	3.91	3.94	3.98	3.92	1.23


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,4	Ac		23.3-23.4/2.05-2.06
3,4	aLRhap4N	103.0/5.17	70.3/4.20	78.0/3.97	52.5/3.97	69.4/3.92	18.0/1.23
3,2	Ac		23.3-23.4/2.05-2.06
3	aDGalpN	95.0/5.04	50.9/4.26	69.2/3.97	77.2/4.12	72.7/3.90	62.2/3.74-3.81
4	Ac		23.3-23.4/2.05-2.06
	aLRhap4N	103.0/5.00	67.0/3.91	74.3/3.94	52.3/3.98	69.4/3.92	18.0/1.23

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

577.584 g/mol (C₂₄H₃₉R₂N₃O₁₃, R = next and previous repeats, not counted in mol weight)