Little is known about the underlying basis of serotype specificity among strains of Flavobacterium psychrophilum, the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the O-polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of Fp CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of Fp 950106-1/1, which belongs to a different O-serotype, are identical except for wzy, which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for Fp CSF259-93 versus β1-3 for Fp 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti-F. psychrophilum antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of Fp CSF259-93 or Fp 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.

Lipopolysaccharide, O-Polysaccharide structure, Flavobacterium psychrophilum, Fish pathogen, O-polysaccharide genes, O-serotypes

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2	Ac	?	23.01
3,3	aLFucpN	97.79	50.20	67.81	81.36	67.92	16.84
3,2	Ac	?	23.14
3,4	lX3,5HOHex	?	44.83	67.60	45.98	66.34	22.93
3	bDQuipN4N	103.28	57.38	76.14	56.34	71.52	18.05
	aLRhap	102.48	71.07	80.57	72.01	70.55	17.41


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	Ac	-	1.963
3,3	aLFucpN	5.106	4.176	3.825	3.844	3.947	1.243
3,2	Ac	-	2.053
3,4	lX3,5HOHex	-	2.419	4.157	1.617-1.738	3.969	1.212
3	bDQuipN4N	4.679	3.939	3.858	3.764	3.630	1.248
	aLRhap	4.818	4.232	3.885	3.503	4.027	1.214


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	Ac		23.01/1.963
3,3	aLFucpN	97.79/5.106	50.20/4.176	67.81/3.825	81.36/3.844	67.92/3.947	16.84/1.243
3,2	Ac		23.14/2.053
3,4	lX3,5HOHex		44.83/2.419	67.60/4.157	45.98/1.617-1.738	66.34/3.969	22.93/1.212
3	bDQuipN4N	103.28/4.679	57.38/3.939	76.14/3.858	56.34/3.764	71.52/3.630	18.05/1.248
	aLRhap	102.48/4.818	71.07/4.232	80.57/3.885	72.01/3.503	70.55/4.027	17.41/1.214

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

649.691 g/mol (C₂₈H₄₇R₂N₃O₁₄, R = next and previous repeats, not counted in mol weight)

Little is known about the underlying basis of serotype specificity among strains of Flavobacterium psychrophilum, the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the O-polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of Fp CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of Fp 950106-1/1, which belongs to a different O-serotype, are identical except for wzy, which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for Fp CSF259-93 versus β1-3 for Fp 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti-F. psychrophilum antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of Fp CSF259-93 or Fp 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.

Lipopolysaccharide, O-Polysaccharide structure, Flavobacterium psychrophilum, Fish pathogen, O-polysaccharide genes, O-serotypes

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,3,2	Ac	?	23.01
2,3	aLFucpN	97.79	50.20	67.81	81.36	67.92	16.84
2,2	Ac	?	23.14
2,4	lX3,5HOHex	?	44.86	67.61	46.01	66.34	22.91
2	aDQuipN4N	96.38	55.35	73.69	56.07	67.73	17.67
	aLRhap	99.77	76.72	70.48	72.87	70.48	17.46


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,3,2	Ac	-	1.963
2,3	aLFucpN	5.113	4.131	3.820	3.825	3.917	1.214
2,2	Ac	-	2.053
2,4	lX3,5HOHex	-	2.419	4.157	1.622-1.738	3.969	1.212
2	aDQuipN4N	4.838	4.160	3.957	3.786	4.147	1.207
	aLRhap	4.768	4.036	3.975	3.525	4.062	1.247


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,3,2	Ac		23.01/1.963
2,3	aLFucpN	97.79/5.113	50.20/4.131	67.81/3.820	81.36/3.825	67.92/3.917	16.84/1.214
2,2	Ac		23.14/2.053
2,4	lX3,5HOHex		44.86/2.419	67.61/4.157	46.01/1.622-1.738	66.34/3.969	22.91/1.212
2	aDQuipN4N	96.38/4.838	55.35/4.160	73.69/3.957	56.07/3.786	67.73/4.147	17.67/1.207
	aLRhap	99.77/4.768	76.72/4.036	70.48/3.975	72.87/3.525	70.48/4.062	17.46/1.247

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

649.691 g/mol (C₂₈H₄₇R₂N₃O₁₄, R = next and previous repeats, not counted in mol weight)