Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd

Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influ enzae, strain Rd, a capsule-deficient type d strain, has been achieved by u sing high-field NMR techniques and electrospray ionization-mass spectrometr y (ESI-MS) on delipidated LPS and core oligosaccharide samples. It was foun d that this organism expresses heterogeneous populations of LPS of which th e oligosaccharide (OS) epitopes are subject to phase variation. ESI-MS of O -deacylated LPS revealed a series of related structures differing in the nu mber of hexose residues linked to a conserved inner-core element, L-alpha-D -Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp- (1-->5)-alpha-Kdo, and the degree of phosphorylation, The structures of the major LPS glycoforms containing three (two Glc and one Gal), four (two Glc and two Gal) and five (two Glc, two Gal and one GalNAc) hexoses were subst ituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and wer e determined in detail. In the major glycoform, Hex3, a lactose unit, beta- D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner-core element. The Hex4 glycoform contains the P-K epi tope, alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp while in the Hex 5 glycoform, this OS is elongated by the addition of a terminal beta-D-Galp NAc residue, giving the P antigen, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-- >4)-beta-D-Galp-(1-->4)-D-Glcp. The fully extended LPS glycoform (Hex5) has the following structure. [GRAPHICS] beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp -(1-->2)-L-alpha-D-Hepp The structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. infl uenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd(-) derived mutant strain (RM .118-28) [Risberg, A., Schweda, E. K, H. Br Jansson, P-E. (1997) Eur J. Bio chem, 243, 701-707].