Genes of non-typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines.
Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.
van Ulsen
Peter
P
Laboratory for Vaccine Research, National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands. P.vanUlsen@bio.uu.nl
van Schilfgaarde
Muriel
M
Dankert
Jacob
J
Jansen
Henk
H
van Alphen
Loek
L
D000222:Adaptation, Physiological; D018277:Carcinoma, Mucoepidermoid / Q000473:pathology; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004847:Epithelial Cells / Q000382:microbiology*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006193:Haemophilus influenzae / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured / Q000382:microbiology
D004269:DNA, Bacterial; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase