An Alternate Pattern for Globoside Oligosaccharide Expression in Haemophilus influenzae Lipopolysaccharide: Structural Diversity in Nontypeable Strain 1124.

Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [β-D-GalpNAc- (1→3)-α-D-Galp-( 1→4)-β-D-Galp-( 1→4) -β-D-Glcp] and its truncated versions globo side [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) of the triheptosyl inner-core moiety L-α-D-Hepp-(1→2)-[PEA→6]L-α-D-Hepp-(1→3)- L-α-D-HepP-(1→5)-[PPEA→4]-α..Kdo(2→6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and Hepill at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of α-D-Galp-(1→4)-β -D-G alp in both extensions, whereas lic2A directs only the expression of β-D-Galp-(1→4)-β-D-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MSIMS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphorylcholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII. [ABSTRACT FROM AUTHOR]