Structural characterization of Haemophilus parainfluenzae lipooligosaccharide and elucidation of its role in adherence using an outer core mutant.

ABSTRACT

The opportunistic pathogen Haemophilus parainfluenzae is a gram-negative bacterium found in the oropharynx of humans. Haemophilus parainfluenzae is a member of the Pasteurellaceae family in which it is most closely related to Haemophilus sengis and Actinobacillus. Characterization of surface displayed lipooligosaccharide has identified components that are crucial in adherence. We examined the oligosaccharide structure of lipooligosaccharide from 2 clinical isolates of H. parainfluenzae. Core oligosaccharide was isolated by standard methods from purified lipooligosaccharide. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy, and mass spectrometry revealing the following structures R-(1-6)-beta-Glc-(1-4)-D,D-alpha-Hep-(1-6)-beta-Glc-(1-4)- substituting a tri-heptose-Kdo inner core of L,D-alpha-Hep-(1-2)-L,D-alpha-Hep-(1-3)-L,D-alpha-Hep-(1-5)-alpha-Kdo at the 4-position of the proximal L,D-alpha-Hep residue to Kdo, and with a PEtn residue at the 6-position of the central L,D-alpha-Hep residue. In strain 4282, the R substituent is beta-galactose and in strain 4201 there is no substituent at the distal glucose. These analyses have revealed that multiple structural aspects of H. parainfluenzae lipooligosaccharide are comparable with nontypeable Haemophilus influenzae lipooligosaccharide. This study also identified a galactan in strain 4201 and a glucan in strain 4282. Haemophilus parainfluenzae was shown to adhere to a bronchial epithelial cell line to the same degree as nontypeable H. influenzae. However, an H. parainfluenzae mutant lacking the outer core of the lipooligosaccharide showed diminished adherence to the epithelial cells, suggesting that H. parainfluenzae lipooligosaccharide plays a role in tissue colonization.