Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-α-D-GalNAcAN-(1→4)-α-D-GalNAcAN-(1→3)-β-D-QuiNAc-(1→2)-β-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

Pathogenesis, O-antigen, capsular polysaccharide, Francisella tularensis, immunogenicity

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,3,4,2	Ac
2,3,4,6	NH2
2,3,4	aDGalpNA	100.1	53.7	73.1	82.2	72.8	?
2,3,2	Ac
2,3,6	NH2
2,3	aDGalpNA	100.9	51.8	70	78	70.1	?
2,2	Ac
2	bDQuipN	104.4	57.5	82.4	78.9	74.4	18.9
4	Fo
	bDQuip4N	105.3	83	76.8	58.5	73.3	19.5


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,3,4,2	Ac
2,3,4,6	NH2
2,3,4	aDGalpNA	5.09	4.07	4.26	4.51	4.86	-
2,3,2	Ac
2,3,6	NH2
2,3	aDGalpNA	5.4	4.24	4.02	4.44	4.04	-
2,2	Ac
2	bDQuipN	4.75	3.72	3.66	3.34	3.44	1.2
4	Fo
	bDQuip4N	4.41	3.53	3.52	3.62	3.43	1.19


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,3,4,2	Ac
2,3,4,6	NH2
2,3,4	aDGalpNA	100.1/5.09	53.7/4.07	73.1/4.26	82.2/4.51	72.8/4.86	
2,3,2	Ac
2,3,6	NH2
2,3	aDGalpNA	100.9/5.4	51.8/4.24	70/4.02	78/4.44	70.1/4.04	
2,2	Ac
2	bDQuipN	104.4/4.75	57.5/3.72	82.4/3.66	78.9/3.34	74.4/3.44	18.9/1.2
4	Fo
	bDQuip4N	105.3/4.41	83/3.53	76.8/3.52	58.5/3.62	73.3/3.43	19.5/1.19

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

792.749 g/mol (C₃₁H₄₈R₂N₆O₁₈, R = next and previous repeats, not counted in mol weight)