Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is capable of expressing a heterogeneous population of LPS which exhibits extensive antigenic diversity among multiple oligosaccharide (OS) epitopes. Structural elucidation of variable and conserved OS epitopes of H. influenzae serotype b strain Eagan was determined by the application of high-field NMR techniques and MS-based methods on oligosaccharides obtained from LPS samples by a deacylation strategy. LPS extracted by the hot aqueous phenol method gave complex electrophoretic patterns consisting of at least six low-molecular mass bands. Electrospray ionization-mass spectrometry of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues as well as subpopulations of glycoforms containing additional phosphoethanolamine (PEA) groups. It was demonstrated that the LPS contains a conserved PEA-substituted, heptose-containing trisaccharide inner core moiety attached via a KDO 4-phosphate unit to a lipid A component. Tandem MS experiments unambiguously established the presence of a KDO 4-pyrophosphoethanolamine unit in the subpopulation of LPS containing additional PEA groups. The occurrence of LPS containing this structural feature was found to be dependant on the isolation procedure used. Each heptose of the common inner core element L-alpha-D-Hepp(1-->2)-L-alpha-D-Hepp(1-->3)-L-alpha-D-Hepp(1-->5)-alpha-KDO is substituted by a hexose residue with further chain elongation from the central unit. The structures of the major glycoforms containing four (three Glcs and one Gal), five (three Glcs and two Gals), and six (three Glcs and three Gals) hexoses were determined in detail. The Hex6 glycoform contains the terminal structure, alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glc, providing, for the first time, definitive structural evidence for the expression of the pk-blood group antigen in H. influenzae LPS. Moreover, an analogue of the Hex4 glycoform was identified in which the third heptose residue carries phosphate at O-4.