E.K.H. Schweda <Elke.Schweda
The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [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] with PCho→6)-β-D-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses 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-(1→] and its truncated versions globoside [α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→] and lactose [β-D-Galp-(1→4)-β-D-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-D-Gal-(1→4)-β-D-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.
13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, HF solvolysis, ESI-MS, mild acid hydrolysis, de-O-acylation with hydrazine, composition analysis, NMR-1D, alkaline deacylation, HPAEC-PAD, CE-ESI-MS/MS, CE-ESI-MS, LC-ESI-MS
Hex6 glycoform core oligosaccharide from H. influenzae (NTHi) 1008; signal 4 (72.2) in the #5,4,4,4_bDGalp 13C NMR spectrum was replaced by ? due to NMR simulation
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7
5,3,2,2,4 bDGalp 103.6 71.3 72.3 72.4 ? ?
5,3,2,2 bDGlcp 102.7 73.2 73.9 78.8 75.3 60.9
5,3,2 aXLDmanHepp 100.4 78.7 ? ? ? ? ?
5,3,6,0 xXEtN
5,3,6 P
5,3 aXLDmanHepp 99.4 79.1 ? ? ? 74.0 ?
5,4,4,4,4 aDGalp 100.6 68.9 69.7 70.1 ? ?
5,4,4,4 bDGalp 103.6 71.4 70.9 ? ? ?
5,4,4 bDGlcp 102.2 73.3 75.4 80.6 75.4 61.0
5,4,6,0 xXCho
5,4,6 P
5,4 bDGlcp 103.2 73.2 75.3 79.7 73.9 64.6
5 aXLDmanHepp 97.0 71.9 ? 74.6 ? ? ?
4,0,0 xXEtN
4,0 %xXP?
4 P
aXKdop
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7
5,3,2,2,4 bDGalp 4.47 3.56 3.68 3.94 ? ?
5,3,2,2 bDGlcp 4.67 3.33 3.68 3.65 3.65 3.84-3.99
5,3,2 aXLDmanHepp 5.07 4.20 4.04 3.93 ? ? ?
5,3,6,0 xXEtN
5,3,6 P
5,3 aXLDmanHepp 5.56 4.25 3.97 ? 3.87 4.54 3.74
5,4,4,4,4 aDGalp 4.93 3.84 3.90 4.03 ? ?
5,4,4,4 bDGalp 4.44 3.54 3.67 3.95 ? ?
5,4,4 bDGlcp 4.83 3.30 3.67 3.72 3.67 3.84-3.98
5,4,6,0 xXCho
5,4,6 P
5,4 bDGlcp 4.52 3.43 3.64 3.74 3.67 4.18-4.34
5 aXLDmanHepp 5.02 3.67 3.92 4.19 ? 4.19 ?
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
5,3,2,2,4 bDGalp 103.6/4.47 71.3/3.56 72.3/3.68 72.4/3.94 ?/? ?/?
5,3,2,2 bDGlcp 102.7/4.67 73.2/3.33 73.9/3.68 78.8/3.65 75.3/3.65 60.9/3.84-3.99
5,3,2 aXLDmanHepp 100.4/5.07 78.7/4.20 ?/4.04 ?/3.93 ?/? ?/? ?/?
5,3,6,0 xXEtN
5,3,6 P
5,3 aXLDmanHepp 99.4/5.56 79.1/4.25 ?/3.97 ?/? ?/3.87 74.0/4.54 ?/3.74
5,4,4,4,4 aDGalp 100.6/4.93 68.9/3.84 69.7/3.90 70.1/4.03 ?/? ?/?
5,4,4,4 bDGalp 103.6/4.44 71.4/3.54 70.9/3.67 ?/3.95 ?/? ?/?
5,4,4 bDGlcp 102.2/4.83 73.3/3.30 75.4/3.67 80.6/3.72 75.4/3.67 61.0/3.84-3.98
5,4,6,0 xXCho
5,4,6 P
5,4 bDGlcp 103.2/4.52 73.2/3.43 75.3/3.64 79.7/3.74 73.9/3.67 64.6/4.18-4.34
5 aXLDmanHepp 97.0/5.02 71.9/3.67 ?/3.92 74.6/4.19 ?/? ?/4.19 ?/?
4,0,0 xXEtN
4,0 %xXP?
4 P
aXKdop
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Displayed record 1
, Life stage: adult]; chronic respiratory tract infections [ICD11: CA45 
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