Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, L-alpha-d-Hepp -(1-->2)-[P Etn-->6]-L-alpha-d-Hepp -(1-->3)-[beta-d-Glcxp-(1-->4)]-L-alpha-d-Hepp -(1-->5)-[PP Etn-->4]-alpha-Kdop -(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by P Cho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-Galp NAc-(1-->3)-alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a P Etn group was identified at O-3 of the distal heptose residue in the inner-core.
Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media and respiratory tract infections. Its lipopolysaccharide (LPS) molecule, an outer membrane component, is a major virulence factor of NTHi. The LPS molecule may also be an efficient target for antibodies which might be protective against NTHi disease. The present thesis describes the structural analyses of the LPS from a number of NTHi clinical isolates. These isolates have been selected to be representative of the genetic diversity in the natural population of NTHi. The studies revealed extended inter- and intrastrain variability in LPS expression but also the presence of a conserved structural element. Several novel structural features were found, including epitopes not previously observed in Haemophilus influenzae LPS. The studies were performed using nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry and different chemical degradations of the LPS as the principal methods.