We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 2019, a prototype strain that is used for studies of NTHi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS(n) on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, 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. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→3 and truncated versions thereof. PEtn substitutes O-3 of the distal heptose (HepIII) of the inner-core moiety. This PEtn substituent was absent in the lpt3 mutant indicating that Lpt3 is the transferase required to add PEtn to the distal heptose. Interestingly, in the lex2B mutant strain HepIII was found to be substituted at O-2 by β-D-Glcp which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having β-D-Glcp linked to O-4 of HepI and also glycoforms with an additional PEtn which could be assigned to HepIII. Acetate groups and one glycine residue further substitute HepIII in NTHi 2019.
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