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-α-d-Hepp(1→2)-l-α-d-Hepp(1→3)-l-α-d-Hepp(1→5)-α-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, α-d-Galp(1→4)-β-d-Galp(1→4)-β-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.

Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influenzae, strain Rd, a capsule-deficient type d strain, has been achieved by using high-field NMR techniques and electrospray ionization-mass spectrometry (ESI-MS) on delipidated LPS and core oligosaccharide samples. It was found that this organism expresses heterogeneous populations of LPS of which the oligosaccharide (OS) epitopes are subject to phase variation. ESI-MS of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues linked to a conserved inner-core element,, 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 substituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and were determined in detail. In the major glycoform, Hex3, a lactose unit, b-d-Galp- (134) The fully extended LPS glycoform (Hex5) has the following structure.PPEtnThe structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. influenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd 2 derived mutant strain (RM

The availability of the complete 1.83-megabase-pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H.influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.