Acylation of the lipooligosaccharide of Haemophilus influenzae and colonization: an htrB mutation diminishes the colonization of human airway epithelial cells

Abstract

Haemophilus influenzae is a commensal and opportunistic pathogen of the human airways. A number of surface molecules contribute to colonization of the airways by H. influenzae, such as adhesins, including structures found in the lipooligosaccharide (LOS). A human bronchiolar xenograft model was employed to investigate the host-bacterial interactions involved in the colonization of the airway by H. influenzae. Differential display was used to identify H. influenzae mRNA that reflect genes which were preferentially expressed in the xenograft compared to growth. Eleven mRNA fragments had consistent increased expression when the bacteria grew in xenografts. On sequencing these fragments, eight open reading frames were identified. Three of these had no match in the NCBI or the TIGR database, while an additional three were homologous to genes involved in heme or iron acquisition and utilization: two of the mRNAs encoded proteins homologous to enzymes involved in LOS biosynthesis: a heptosyl transferase (rfaF) involved in the synthesis of the LOS core and a ketodeoxyoctonate phosphate-dependent acyltransferase (htrB) that performs one of the late acylation reactions in lipid A synthesis. Inoculation of human bronchiolar xenografts revealed a significant reduction in colonization capacity by htrB mutants. In vitro, htrB mutants elicited lesser degrees of cytoskeletal rearrangement and less stimulation of host cell signaling with 16HBE14o(-) cells and decreased intracellular survival. These results implicate acylation of H. influenzae lipid A as playing a key role in the organisms' colonization of the normal airway.

FIG. 1.

Cryosection of human bronchial epithelial cell xenograft infected with the htrB mutant B29. Bacteria were visualized by immunohistochemical staining with monoclonal antibody 3B9 (IgG), which recognizes the H. influenzae outer membrane protein P6 (A) and monoclonal antibody TEPC-15 (IgA), which recognizes ChoP (B). Goat anti-mouse IgG-FITC (Molecular Probes) and anti-mouse IgA-Texas red conjugate (Cortex Biochemical) secondary antibodies were used to identify bacteria (arrows). (C) Epithelial cell monolayer as visualized by Nomarski differential-interference contrast microscopy. (D) Image in which panels A and B are merged, with the resultant color (faint yellow) photographing as white (arrows). Comparable results were obtained in similar analyses of cells infected with strain 2019 (data not shown).

FIG. 2.

Analysis of immortalized 16HBE14o⁻ human airway epithelial cells infected with NTHi 2019 (A, C, E, and G) and the isogenic NTHi 2019 htrB mutant B29 (B, D, F, and H). The airway cells were cultured at an air-fluid interface on BioCoat semipermeable membrane inserts (1-μm pore size), infected for 4 h as indicated, and processed for SEM analysis (A and B) or embedded in Epon resin, sectioned, and stained for TEM analysis (C to H). Note the difference in microvillus extension in airway cells infected with strain 2019 (A) compared to B29 (B). No apparent differences in the numbers or morphology of intracellular bacteria were observed in TEM analysis of cells infected with strain 2019 (C, E, and G) or B29 (D, F, and H).

FIG. 3.

Effect of inoculation of NTHi 2019 and isogenic mutants on cytosolic IP levels in immortalized 16HBE14o⁻ human airway epithelial cells. Wild-type strain 2019, in comparison with the poorly invasive strain with mutations in pgmB and licD results in the increased cytosolic IP levels by means of PAF receptor activation; mutations affecting the addition of ChoP onto the oligosaccharides of the strain 2019 LOS reduce this effect (43). The 16HBE14o⁻ cells were labeled with ³H-labeled myoinositol and inoculated with the strain indicated, and the IP levels in the cytosolic fractions were assessed.

FIG. 4.

Effect of htrB mutation on ChoP expression by NTHi 2019. Colonies of strain 2019 and the isogenic htrB mutant (strain B29) were lifted onto nitrocellulose, and immunoblot analyses were performed as described in Materials and Methods and in prior studies (42, 47).