Pasteurella multocida is a Gram-negative bacterial pathogen classified into 16 serovars based on lipopolysaccharide (LPS) antigens. Previously, we have characterised the LPS outer core biosynthesis loci L1, L2, L3, L5 and L7, and have elucidated the full range of LPS structures associated with each. In this study, we have determined the LPS structures produced by the type strains representing the serovars 10, 11, 12 and 15 and characterized a new LPS outer core biosynthesis locus, L6, common to all. The L6 outer core biosynthesis locus shares significant synteny with the L3 locus but due to nucleotide divergence, gene duplication, and gene redundancy, the L6 and L3 LPS outer cores are structurally distinct. Using LPS structural and genetic differences identified in each L6 strain we have predicted a role for most of the L6 glycosyltransferases in LPS assembly. Importantly, we have identified two glycosyltransferases, GctD and GatB, that differ by one amino acid, A162T, but use different donor sugars (UDP-Glc and UDP-Gal respectively). The longest outer core oligosaccharide, produced by the serovar 12 type strain, contained a terminal region consisting of β-Gal-(1,4)-β-GlcNAc-(1,3)-β-Gal-(1,4)-β-Glc that was identical in structure to the vertebrate glycosphingolipid, paragloboside. Mimicry of host glycosphingolipids has been observed previously in P. multocida strains belonging to L3 LPS genotype, which produce LPS similar in structure to the globo-series of glycosphingolipids. The expression of a paragloboside-like oligosaccharide on the LPS produced by the serovar 12 type strain indicates that strains belonging to the L6 LPS genotype may also engage in molecular mimicry.

LPS, structure, gene, genetics, core oligosaccharide, glycosyltransferases, glycosyltransferase, Pasteurella multocida, glycosphingolipid

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7
5,3,2	aXLDmanHepp	101.7	70.2	71.0	66.3	71.6	70.6	?
5,3,3,0	xXEtN
5,3,3	P
5,3	aXLDmanHepp	100.3	80.5	75.9	67.2	73.3	70.0	64.4
5,4,6,6	bDGalp	104.3	71.1	72.5	68.6	75.2	?
5,4,6	aXLDmanHepp	101.4	71.0	72.0	67.7	73.1	80.5	64.1
5,4	bDGlcp	104.8	74.9	78.4	70.9	75.2	66.2
5,6	aDGlcp	102.9	73.7	74.5	70.1	73.1	61.0
5	aXLDmanHepp	100.7	71.6	74.7	75.6	73.0	82.0	64.9
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7
5,3,2	aXLDmanHepp	5.21	4.00	3.87	3.82	3.78	4.08	?
5,3,3,0	xXEtN
5,3,3	P
5,3	aXLDmanHepp	5.75	4.32	4.46	4.08	3.67	4.08	3.61-3.72
5,4,6,6	bDGalp	4.48	3.57	3.68	3.93	3.75	?
5,4,6	aXLDmanHepp	5.05	4.04	3.81	3.95	3.90	4.17	3.74-3.90
5,4	bDGlcp	4.59	3.52	3.40	3.61	3.49	3.72-4.07
5,6	aDGlcp	5.21	3.57	3.83	3.56	3.94	3.76-3.96
5	aXLDmanHepp	5.17	4.14	4.02	4.18	4.19	4.07	3.70
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7
5,3,2	aXLDmanHepp	101.7/5.21	70.2/4.00	71.0/3.87	66.3/3.82	71.6/3.78	70.6/4.08	?/?
5,3,3,0	xXEtN
5,3,3	P
5,3	aXLDmanHepp	100.3/5.75	80.5/4.32	75.9/4.46	67.2/4.08	73.3/3.67	70.0/4.08	64.4/3.61-3.72
5,4,6,6	bDGalp	104.3/4.48	71.1/3.57	72.5/3.68	68.6/3.93	75.2/3.75	?/?
5,4,6	aXLDmanHepp	101.4/5.05	71.0/4.04	72.0/3.81	67.7/3.95	73.1/3.90	80.5/4.17	64.1/3.74-3.90
5,4	bDGlcp	104.8/4.59	74.9/3.52	78.4/3.40	70.9/3.61	75.2/3.49	66.2/3.72-4.07
5,6	aDGlcp	102.9/5.21	73.7/3.57	74.5/3.83	70.1/3.56	73.1/3.94	61.0/3.76-3.96
5	aXLDmanHepp	100.7/5.17	71.6/4.14	74.7/4.02	75.6/4.18	73.0/4.19	82.0/4.07	64.9/3.70
4,0,0	xXEtN
4,0	%xXP?
4	P
	aXKdop

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

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

NCCOP(=O)(O)O[C@@H]1[C@H](O[C@H]2O[C@H]([C@@H](O)CO)[C@@H](O)[C@H](O)[C@@H]2O)[C@@H](O[C@@H]2[C@H](O)[C@@H](O[C@@H]3[C@H](OP(=O)(O)OP(=O)(O)OCCN)C[C@](O)(C(=O)O)O[C@@H]3[C@H](O)CO)O[C@H]([C@H](CO)O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@H]2O[C@@H]2O[C@H](CO[C@H]3O[C@H]([C@H](CO)O[C@@H]4O[C@H](CO)[C@H](O)[C@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H](O)[C@H](O)[C@H]2O)O[C@H]([C@@H](O)CO)[C@H]1O

1819.358 g/mol (C₅₈H₁₀₅N₂O₅₆P₃)