Tannerella forsythia is a Gram-negative anaerobic organism that inhabits subgingival plaque biofilms and is covered with a so far unique surface layer composed of two glycoproteins. It belongs to the so-called 'red complex' of bacteria comprising species that are associated with periodontal disease. While the surface layer glycoprotein glycan structure had been elucidated recently and found to be a virulence factor, no structural data on the lipopolysaccharide (LPS) of this organism were available. In this study, the T. forsythia LPS structure was partially elucidated by a combined mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) approach and initial experiments to characterize its immunostimulatory potential were performed. The T. forsythia LPS is a complex, rough-type LPS with a core region composed of one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue, three mannose residues, and two glucosamine residues. MS analyses of O-deacylated LPS proved that, in addition, one phosphoethanolamine residue and most likely one galactose-phosphate residue were present, however, their positions could not be identified. Stimulation of human macrophages with T. forsythia LPS resulted in the production of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in a dose-dependent manner. The response to T. forsythia LPS was observed only upon stimulation in the presence of fetal calf serum (FCS), whereas no cytokine production was observed in the absence of FCS. This finding suggests that the presence of certain additional cofactors is crucial for the immune response induced by T. forsythia LPS.

Lipopolysaccharide, structure, Kdo, core region, Biofilm, cytokines, Tannerella forsythia

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
6,6,5,3,6	aDGlcpN	96.8	55.3	71.5	70.6	73.4	61.5
6,6,5,3	aDManp	101.0	71.1	71.3	67.3	72.2	66.7
6,6,5,6,6	aDGlcpN	96.7	55.3	71.4	70.6	73.4	61.5
6,6,5,6	aDManp	104.0	71.2	72.1	67.1	72.1	66.4
6,6,5	aDManp	102.7	71.3	80.1	66.8	72.6	66.6
6,6	aXKdop	175.7	101.0	36.1	67.1	76.8	72.8	70.7	64.7
6	bDGlcpN	102.8	57.3	75.2	71.2	75.6	62.9
	xDGlcN-ol	60.1	56.5	67.1	70.5	71.8	73.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
6,6,5,3,6	aDGlcpN	5.15	3.29	3.90	3.50	3.77	3.81-3.88
6,6,5,3	aDManp	4.90	4.02	3.85	3.84	3.82	3.71-4.10
6,6,5,6,6	aDGlcpN	5.23	3.37	3.92	3.52	3.77	3.81-3.88
6,6,5,6	aDManp	5.14	4.11	3.94	3.94	3.67	3.68-4.20
6,6,5	aDManp	5.04	4.16	3.99	3.92	4.22	3.65-4.00
6,6	aXKdop	-	-	1.87-2.13	4.16	4.13	3.70	3.84	3.66-3.96
6	bDGlcpN	4.60	2.88	3.51	3.49	3.62	3.59-3.62
	xDGlcN-ol	3.77-3.87	3.53	4.11	3.95	3.89	3.82-4.16


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
6,6,5,3,6	aDGlcpN	96.8/5.15	55.3/3.29	71.5/3.90	70.6/3.50	73.4/3.77	61.5/3.81-3.88
6,6,5,3	aDManp	101.0/4.90	71.1/4.02	71.3/3.85	67.3/3.84	72.2/3.82	66.7/3.71-4.10
6,6,5,6,6	aDGlcpN	96.7/5.23	55.3/3.37	71.4/3.92	70.6/3.52	73.4/3.77	61.5/3.81-3.88
6,6,5,6	aDManp	104.0/5.14	71.2/4.11	72.1/3.94	67.1/3.94	72.1/3.67	66.4/3.68-4.20
6,6,5	aDManp	102.7/5.04	71.3/4.16	80.1/3.99	66.8/3.92	72.6/4.22	66.6/3.65-4.00
6,6	aXKdop			36.1/1.87-2.13	67.1/4.16	76.8/4.13	72.8/3.70	70.7/3.84	64.7/3.66-3.96
6	bDGlcpN	102.8/4.60	57.3/2.88	75.2/3.51	71.2/3.49	75.6/3.62	62.9/3.59-3.62
	xDGlcN-ol	60.1/3.77-3.87	56.5/3.53	67.1/4.11	70.5/3.95	71.8/3.89	73.0/3.82-4.16

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

N[C@@H](CO)[C@@H](O)[C@H](O)[C@H](O)CO[C@@H]1O[C@H](CO[C@]2(C(=O)O)C[C@@H](O)[C@@H](O[C@H]3O[C@H](CO[C@H]4O[C@H](CO[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5N)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O[C@H]4O[C@H](CO[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5N)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H]3O)[C@@H]([C@H](O)CO)O2)[C@@H](O)[C@H](O)[C@H]1N

1371.259 g/mol (C₅₀H₉₀N₄O₃₉)