Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae.

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)-ß-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [ß-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-ß-d-Galp-(1→4)-ß-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-ß-d-Galp-(1→4)-ß-d-Glcp-(1→] and lactose [ß-d-Galp-(1→4)-ß-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)-ß-d-Gal-(1→4)-ß-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.