A novel branching pattern in the lipopolysaccharide expressed by non-typeable Haemophilus influenzae strain 1232

Abstract

We report the novel branching pattern in lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strain 1232. The strain expressed the β-d-Glcp-(1→4)-[α-d-Galp-(1→4)-β-d-Galp-(1→7)]-d-α-d-Hepp-(1→6)-β-d-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. The structure has been elucidated using NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MS(n)) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS(n) on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [α-Neu5Ac-(2→3)-β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-(1→] could substitute O-4 of the β-d-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho→6-β-d-Galp-(1→ at the O-2 position.

Keywords: 3-deoxy-d-manno-oct-2-ulosonic acid; AnKdo-ol; CE; ESI-MS; Haemophilus influenzae; Hex; HexNAc; Kdo; LPS; LPS-OH; Lipopolysaccharide; MS(n); N-acetylhexosamine; N-acetylneuraminic acid, sialic acid; NTHi; Neu5Ac; O-deacylated lipopolysaccharide; OS; P; PCho; PEtn; PPEtn; Sialyllacto-N-neotetraose; capillary electrophoresis; d,d-Hep; d-glycero-d-manno-Heptose; d-glycero-d-manno-heptose; electrospray ionization mass spectrometry; hexose; l,d-Hep; l-glycero-d-manno-heptose; lipopolysaccharide; losA1; multiple step tandem mass spectrometry; non-typeable Haemophilus influenzae; oligosaccharide; phosphate; phosphocholine; phosphoethanolamine; pyrophosphoethanolamine; reduced anhydro Kdo.