Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp -(1-->2)-[P Etn-->6]-l-alpha-d-Hepp -(1-->3)-[beta-d-Glcp -(1-->4)]-l-alpha-d-Hepp -(1-->5)-[PP Etn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glcp -(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp -(1-->4)-alpha-d-Glcp ). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.

Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MSn). All of the analyzed strains showed a major lipid A molecule comprising beta-2-amino-2-deoxy-D-glucopyranose-(1 -> 6)-alpha-2-amino-2-deoxy-D-glucopyranose phosphorylated at the C4 ' and C1 positions. The C2/C2 ' and C3/C3 ' positions were substituted by amide-linked and esterlinked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains oil C3 ' and C2 ' were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being Substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3 ' positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.

Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [beta-D-GalpNAc-(1 -> 3)-alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and its truncated versions globoside [alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and lactose [beta-D-Galp-(1 -> 4)-beta-D-Glcp] linked to the tenninal heptose (HepIII) of the triheptosyl inner-core moiety L-alpha-D-Hepp-(1 -> 2)-[PEA -> 6]-L-alpha-D-Hepp-(1 -> 3)L-alpha-D-Hepp-(1 -> 5)-[PPEA -> 4]-alpha-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 HepIII 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 beta-D-Galp-(1 -> 4)-beta-D-Galp in both extensions, whereas lic2A directs only the expression Of beta-D-Ga1p-(1 -> 4)-beta-D-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. 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 phosphoryleholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII.

The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy 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. It was found that the LPS contains the common structural element of H. influenzae, L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glcp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-[PPEtn -> 4]-alpha-Kdo-(2 -> 6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEW at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1 -> 4)-beta-D-Glcp-(1 -> or beta-D-Glcp-(1 ->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MS" in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.