The structural diversity of lipopolysaccharide expressed by non-typeable Haemophilus influenzae strains 1158 and 1159

A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS(n) on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-D-α-D-Hepp-(1→6)-β-D-Glcp linked to HepI from the common structural element of H. influenzae 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. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of β-D-Glcp attached to HepIII and was also located at a novel position linked to D-α-D-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MS(n) experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the D,D-Hep to the outer core region in strain 1158.