The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-D-Glcp-(1→4)-α-Kdop-(2→4)[β-D-GlcpN-(1→6)-β-D-GlcpN-(1→4) [β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp-(1→5)]-α-Kdop-(2→6)-β-D-GlcpN3N4P-(1→6)-α-D-GlcpN3N1P), in addition to components lacking one of the terminal β-D-GlcpN and/or the β-D-Glcp residue (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep rough pentasaccharide core (β-D-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-D-GlcpN3N4P-(1→ 6)-α-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-D-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyl transferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the [β-D-GlcpN-(1→6)-β-D-GlcpN-(1→4)[β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp-(1→5)] structure in virulence.

mutant, Gram-negative bacteria, Brucella melitensis, lipopolysaccharide (LPS), nuclear magnetic resonance (NMR), WadC

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
0,6,6,4,4	63%bDGlcp	102.23	73.84	76.36	70.39	76.59	61.40
0,6,6,4	aXKdop	177.40	100.15	33.13	75.63	66.60	73.25	70.55	64.16
0,6,6	aXKdop	175.75	100.46	34.20	69.45	65.42	72.15	70.65	64.11
0,6,4	P
0,6	bDGlcpN3N	103.87	55.47	58.60	71.05	76.41	63.40
0	aDGlcpN3N	94.08	54.59	54.97	69.27	72.43	70.23
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
0,6,6,4,4	63%bDGlcp	4.632	3.296	3.521	3.418	3.485	3.737-3.918
0,6,6,4	aXKdop	-	-	1.846-2.286	4.237	4.270	3.626	3.983	3.754-4.016
0,6,6	aXKdop	-	-	1.940-2.002	4.156	4.096	3.738	3.897	3.689-3.899
0,6,4	P
0,6	bDGlcpN3N	4.577	2.786	3.172	3.797	3.730	3.481-3.804
0	aDGlcpN3N	5.430	2.955	3.171	3.508	4.128	3.742-4.263
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
0,6,6,4,4	63%bDGlcp	102.23/4.632	73.84/3.296	76.36/3.521	70.39/3.418	76.59/3.485	61.40/3.737-3.918
0,6,6,4	aXKdop			33.13/1.846-2.286	75.63/4.237	66.60/4.270	73.25/3.626	70.55/3.983	64.16/3.754-4.016
0,6,6	aXKdop			34.20/1.940-2.002	69.45/4.156	65.42/4.096	72.15/3.738	70.65/3.897	64.11/3.689-3.899
0,6,4	P
0,6	bDGlcpN3N	103.87/4.577	55.47/2.786	58.60/3.172	71.05/3.797	76.41/3.730	63.40/3.481-3.804
0	aDGlcpN3N	94.08/5.430	54.59/2.955	54.97/3.171	69.27/3.508	72.43/4.128	70.23/3.742-4.263
	P

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]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@@H]([C@H](O)CO)O4)[C@@H](O)[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)