Anew structural type for Haemophilus influenzae lipopolysaccharide -: Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common 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 -Kdop- (2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha -Neu5Ac(2-->3)-beta -D-Galp-(1-->4)-beta -D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha -D-Glcp, residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.