Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. NTHi releases lipooligosaccharide (LOS) as outer membrane fragments during its growth. The release of LOS may play an important role in the pathogenicity of otitis media caused by this organism. The amounts of LOS in bacterial cells and growth media for five NTHi strains were determined by quantitative silver staining after sodium dodecyl sulfate- polyacrylamide gel electrophoresis. These strains were estimated to have 1.6 x 10⁶ to 4.8 x 10⁶ LOS molecules per bacterium. During a 3-day growth period, these NTHi strains released variable but significant amounts of LOS into the growth medium. Cells started to release detectable amounts of LOS into the medium at 2 to 5 h and continued to do so for up to 48 or 72 h. The concentrations of LOS in the culture supernatants released by these five strains were 10 to 55 μg/ml at 24 h and 40 to 100 μg/ml at 72 h, which was 34 to 189% of the cell-bound LOS concentration. The biological properties of released and cell-bound LOSs from two representative strains were compared. Released LOS showed an approximately 10-fold increase in inducing human monocytes to produce tumor necrosis factor alpha, interleukin 1β, and interleukin 6, a 13- to 28-told increase in mouse lethal toxicity, and a 16- to 37-told increase in the clotting of Limulus amebocyte lysate. These results suggested that released LOS or its inflammatory mediators play a more important role than the LOS in bacteria in the pathogenicity of otitis media caused by this organism.