Applications of Combined Capillary Electrophoresis-Electrospray Mass Spectrometry in the Characterization of Short-Chain Lipopolysaccharides: Haemophi

Lipopolysaccharide (LPS) is an essential component of the outer membrane of all Gram-negative bacteria (1 ). This complex class of lipoglycans can trigger a cascade of immunological responses in mammals including endotoxic effects and serum antibody production. LPSs have been found to exhibit a common molecular architecture consisting of at least two distinct regions: a carbohydrate-containing region and a lipid moiety referred to as lipid A (2 ). In enteric bacteria (e.g., Escherichia coli, Salmonella strains), the carbohydrate-containing region consists of a high molecular mass O-specific polysaccharide that is covalently linked to a low molecular mass core oligosaccharide (3 ). Other bacteria, including Haemophilus and Neisseria spp., produce only short-chain LPS in which the carbohydrate region typically contains mixtures of low molecular mass but structurally diverse oligosaccharide components (4 ). Short-chain LPS is often referred to as lipooligosaccharide (LOS).Haemophilus influenzae expresses heterogeneous populations of these low molecular mass LPSs which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. This pathogen remains a major cause of disease worldwide. Six capsular serotypes and an indeterminate number of nontypable (i.e., acapsular) strains ofH. influenzae are recognized. In the developed world, nontypable strains are the second major cause of otitis media infections in children, while serotype b capsular strains are associated with invasive diseases, including meningitis and pneumonia (5 ). The carbohydrate regions ofH. influenzae LPS molecules provide targets for recognition by host immune responses, and expression of certain oligosaccharide epitopes is known to contribute to disease pathogenesis. Molecular structural studies of LPS from a number of different strains has resulted in a structural model in which a conserved *c-glycero-D-manno -heptose (Hep)-containing inner-core trisaccharide moiety is attached via a phosphory lated 3-deoxy-D-manno -oct-2-ulosonic acid (Kdo) residue to the lipid A component (6 –12 ). In this structural model, each of the Hep residues within the triad can provide a point for further oligosaccharide chain elongation. The addition of phosphate-containing substituents, which include free phosphate (P), phosphoethanolamine (PEtn), pyrophosphoethanolamine (PPEtn), and phosphocholine (PCho), also contributes to the structural variability of these molecules. Moreover, H. influenzae LPS can undergo phase variation between defined oligosaccharide structures, which creates the possibility of an extensive repertoire of oligosaccharide epitopes in a single strain (13 ,14 ). The structural diversity arising from phase variation has complicated the study of the molecular features of these molecules and their role in commensal and pathogenic behavior in the host. The availability of the complete sequence of the H. influenzae strain Rd genome (15 ) has led to significant progress in identifying the genes that are responsible for LPS expression in this pathogen (16 ). The heterogeneity and structural complexity of short-chain LPS within and between H. influenzae strains pose significant analytical challenges.