The facultative intracellular pathogen Bartonella henselae is responsible for a broad range of clinical manifestations, including the formation of vascular tumors as a result of increased proliferation and survival of colonized endothelial cells. This remarkable interaction with endotoxin-sensitive endothelial cells and the apparent lack of septic shock are considered to be due to a reduced endotoxic activity of the B. henselae lipopolysaccharide. Here, we show that B. henselae ATCC 49882(T) produces a deep-rough-type lipopolysaccharide devoid of O-chain and report on its complete structure and Toll-like receptor-dependent biological activity. The major short-chain lipopolysaccharide was studied by chemical analyses, electrospray ionization, and matrix-assisted laser desorption/ionization mass spectrometry, as well as by NMR spectroscopy after alkaline deacylation. The carbohydrate portion of the lipopolysaccharide consists of a branched trisaccharide containing a glucose residue attached to position 5 of an α-(2→4)-linked 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide. Lipid A is a pentaacylated β-(1'→6)-linked 2,3-diamino-2,3-dideoxy-glucose disaccharide 1,4'-bisphosphate with two amide-linked residues each of 3-hydroxydodecanoic and 3-hydroxyhexadecanoic acids and one residue of either 25-hydroxyhexacosanoic or 27-hydroxyoctacosanoic acid that is O-linked to the acyl group at position 2'. The lipopolysaccharide studied activated Toll-like receptor 4 signaling only to a low extent (1,000-10,000-fold lower compared with that of Salmonella enterica sv. Friedenau) and did not activate Toll-like receptor 2. Some unusual structural features of the B. henselae lipopolysaccharide, including the presence of a long-chain fatty acid, which are shared by the lipopolysaccharides of other bacteria causing chronic intracellular infections (e.g. Legionella and Chlamydia), may provide the molecular basis for low endotoxic potency.

Lipopolysaccharide, structure, structural analysis, biological activity

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
0,6,6,4	aXKdop	?	?	36.1	68.0	68.0	73.6	70.4	64.5
0,6,6,5	aDGlcp	100.8	73.5	73.9	70.5	72.9	61.4
0,6,6	aXKdop	?	?	36.0	73.1	75.1	73.3	72.0	64.0
0,6,4	P
0,6	bDGlcpN3N	105.2	57.5	59.6	75.3	77.5	64.7
0	aDGlcpN3N	95.7	56.7	55.0	71.5	72.8	70.8
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
0,6,6,4	aXKdop	-	-	1.72-2.05	3.90	3.90	3.64	3.96	3.65-3.78
0,6,6,5	aDGlcp	5.22	3.42	3.74	3.46	4.01	3.78-3.78
0,6,6	aXKdop	-	-	1.92-2.02	4.02	4.14	3.68	3.94	3.69-3.85
0,6,4	P
0,6	bDGlcpN3N	4.32	2.48	2.61	3.45	3.57	3.34-3.74
0	aDGlcpN3N	5.26	2.55	2.82	3.26	3.99	3.64-4.18
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
0,6,6,4	aXKdop			36.1/1.72-2.05	68.0/3.90	68.0/3.90	73.6/3.64	70.4/3.96	64.5/3.65-3.78
0,6,6,5	aDGlcp	100.8/5.22	73.5/3.42	73.9/3.74	70.5/3.46	72.9/4.01	61.4/3.78-3.78
0,6,6	aXKdop			36.0/1.92-2.02	73.1/4.02	75.1/4.14	73.3/3.68	72.0/3.94	64.0/3.69-3.85
0,6,4	P
0,6	bDGlcpN3N	105.2/4.32	57.5/2.48	59.6/2.61	75.3/3.45	77.5/3.57	64.7/3.34-3.74
0	aDGlcpN3N	95.7/5.26	56.7/2.55	55.0/2.82	71.5/3.26	72.8/3.99	70.8/3.64-4.18
	P

The spectrum also has 4 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

N[C@H]1[C@H](O)[C@@H](CO[C@@H]2O[C@H](CO[C@]3(C(=O)O)C[C@@H](O[C@]4(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O4)[C@@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)[C@@H]([C@H](O)CO)O3)[C@@H](OP(=O)(O)O)[C@H](N)[C@H]2N)O[C@H](OP(=O)(O)O)[C@@H]1N

1100.814 g/mol (C₃₄H₆₂N₄O₃₂P₂)