Cloning of genes of nontypeable Haemophilus influenzae involved in penetration between human lung epithelial cells

Haemophilus influenzae penetrates between epithelial cells via an unknown m echanism. A chromosomal library of nonencapsulated H. influenzae strain A96 0053 DNA was constructed in Escherichia coli DH5 alpha to identify bacteria l genes contributing to this paracytosis. Two E. coli clones that contained open reading frames (ORFs) homologous to HI0636 to HI0641 of H. influenzae strain Rd and that showed an increased penetration in epithelial cell laye rs of the human bronchial epithelial cell line NCI-H292 were identified. OR Fs HI0636 and HI0638, encoding two small proteins of unknown functions, wer e further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetr ation. Disruption of HI0638 by kanamycin box insertion in H. influenzae str ain A960053 resulted in loss of penetration into the epithelial cell layers . Disruption of HI0636 had no effect on penetration in this model system. S ince a role for HI0637 in the paracytosis of H. influenzae is very unlikely because it encodes TrpS, we conclude that the protein encoded by ORF HI063 8 may function as a paracytin, while that encoded by HI0636 may have an aux iliary function.