Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Huddinge, Sweden
We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [β-D-GalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp] and its truncated versions globoside [α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp] and lactose [β-D-Galp-(1→4)-β-D-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an α-D-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety L-α-D-Hepp-(1→2)-[PEtn→6]-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1 →5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.
13C NMR, methylation, GLC-MS, NMR-2D, HF solvolysis, mild acid hydrolysis, NMR-1D, genetic methods, alkaline hydrolysis, CE-ESI-MS
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7
5,3,2 aXLDmanHepp 102.3 68.4 ? ? ? ? ?
5,3,3,4,4,3,2 Ac ? 23.02
5,3,3,4,4,3 bDGalpN 104.1 53.1 ? ? ? ?
5,3,3,4,4 aDGalp 101.2 68.4 79.8 69.7 ? ?
5,3,3,4 bDGalp 103.2 71.7 72.0 78.0 ? ?
5,3,3,6,0 xXCho 60.1 66.6 ?
5,3,3,6 P
5,3,3 aDGlcp 101.6 72.6 72.0 76.3 71.5 65.1
5,3,6,0 xXEtN 62.7 40.7
5,3,6 P
5,3 aXLDmanHepp 99.0 79.2 80.1 66.8 72.7 75.3 64.1
5,4 bDGlcp 103.4 74.5 76.8 71.3 77.5 63.1
5 aXLDmanHepp 97.5-102.3 70.9 72.9 73.8 ? 68.4 ?
x?Sug?
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
5,3,2 aXLDmanHepp 5.03 3.93 ? ? ? ? ?
5,3,3,4,4,3,2 Ac - 2.05
5,3,3,4,4,3 bDGalpN 4.66 3.97 3.79 3.97 ? ?
5,3,3,4,4 aDGalp 4.92 3.90 3.98 4.28 ? ?
5,3,3,4 bDGalp 4.57-4.64 3.61 3.78 4.05 3.84 ?
5,3,3,6,0 xXCho 4.35 3.29 ?
5,3,3,6 P
5,3,3 aDGlcp 5.28 3.59 3.80 3.80 3.92 4.11-4.18
5,3,6,0 xXEtN 4.14 3.29
5,3,6 P
5,3 aXLDmanHepp 5.83 4.28 4.05 4.14-4.18 3.68 4.59 3.71-3.91
5,4 bDGlcp 4.54 3.46 3.46 3.32 3.46 3.79-3.99
5 aXLDmanHepp 5.05-5.15 3.99-4.05 3.99-4.04 4.31 ? 4.11 ?
x?Sug? - ? 1.87-2.18 ? ? ? ? ?
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
5,3,2 aXLDmanHepp 102.3/5.03 68.4/3.93 ?/? ?/? ?/? ?/? ?/?
5,3,3,4,4,3,2 Ac 23.02/2.05
5,3,3,4,4,3 bDGalpN 104.1/4.66 53.1/3.97 ?/3.79 ?/3.97 ?/? ?/?
5,3,3,4,4 aDGalp 101.2/4.92 68.4/3.90 79.8/3.98 69.7/4.28 ?/? ?/?
5,3,3,4 bDGalp 103.2/4.57-4.64 71.7/3.61 72.0/3.78 78.0/4.05 ?/3.84 ?/?
5,3,3,6,0 xXCho 60.1/4.35 66.6/3.29 ?/?
5,3,3,6 P
5,3,3 aDGlcp 101.6/5.28 72.6/3.59 72.0/3.80 76.3/3.80 71.5/3.92 65.1/4.11-4.18
5,3,6,0 xXEtN 62.7/4.14 40.7/3.29
5,3,6 P
5,3 aXLDmanHepp 99.0/5.83 79.2/4.28 80.1/4.05 66.8/4.14-4.18 72.7/3.68 75.3/4.59 64.1/3.71-3.91
5,4 bDGlcp 103.4/4.54 74.5/3.46 76.8/3.46 71.3/3.32 77.5/3.46 63.1/3.79-3.99
5 aXLDmanHepp 97.5-102.3/5.05-5.15 70.9/3.99-4.05 72.9/3.99-4.04 73.8/4.31 ?/? 68.4/4.11 ?/?
x?Sug? NMR TSV error 2: unequal length of 13C and 1H datasets
report error
Found 1 record.
Displayed record 1
, Life stage: adult]; infection due to Haemophilus influenzae [ICD11: XN1P6 
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