Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae

Haemophilus influenzae expresses heterogeneous populations of short-chain l ipopolysaccharide (LPS) which exhibit extensive antigenic diversity among m ultiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can ca rry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic loc us. The location and site of attachment of PCho substituents were determine d by structural analysis of LPS from two type b H. influenzae strains, Eaga n and RM7004. The lic2 locus is involved in phase variation of oligosacchar ide expression. LPS obtained from the parent strains, from mutants generate d by insertion of antibiotic resistance cassettes in the lic2 genetic locus , and from phase-variants showing high levels of PCho expression was charac terized by electrospray ionization-mass spectrometry (ESI-MS) and H-1 NMR s pectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS fro m wild-type strains revealed mixtures of related glycoform structures diffe ring in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutant s were much less complex than their respective parent strains, consisting o nly of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field N MR techniques. It was found that the LPS samples contain the phosphoethanol amine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-- >6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-alpha-Kdo in which the ma jor glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at th e O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants o f both type b strains were found to carry PCho groups at the O-6 position o f the terminal beta-D-Galp residue attached to HepIII. In the parent strain s, the central heptose (HepII) of the LPS inner-core element is also substi tuted by hexose containing oligosaccharides. The expression of the galabios e epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepI I. From the analysis of core oligosaccharide samples, LPS from the lic2 mut ant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O -acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substitu ted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. Th is is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H . influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 p osition of a beta-D-Glcp, which is in turn attached to HepI.