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

ABSTRACT: Haemophilus influenzae penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated H. influenzae strain A960053 DNA was constructed in Escherichia coli DH5alpha to identify bacterial 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 layers of the human bronchial epithelial cell line NCI-H292 were identified. ORFs HI0636 and HI0638, encoding two small proteins of unknown functions, were further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetration. Disruption of HI0638 by kanamycin box insertion in H. influenzae strain A960053 resulted in loss of penetration into the epithelial cell layers. Disruption of HI0636 had no effect on penetration in this model system. Since 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 HI0638 may function as a paracytin, while that encoded by HI0636 may have an auxiliary function.

SUBMITTER: van Schilfgaarde M

PROVIDER: S-EPMC98391 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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

van Schilfgaarde M M van Ulsen P P van Der Steeg W W Winter V V Eijk P P Everts V V Dankert J J van Alphen L L

Infection and immunity 20000801 8

Haemophilus influenzae penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated H. influenzae strain A960053 DNA was constructed in Escherichia coli DH5alpha to identify bacterial 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 layers of the human bronchial epithelial cell line NCI- ...[more]

PMID: 10899864

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Project description:Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licDI, licDIII, and licDIV) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates.Using dot-blot hybridization, 92% of 88 NT H. influenzae and 42.6% of 109 H. haemolyticus strains possessed a lic1 locus. Eight percent of NT H. influenzae and none of the H. haemolyticus strains possessed dual copies of lic1. The licDIII and licDIV gene alleles were distributed similarly (18-22%) among the NT H. influenzae and H. haemolyticus strains while licDI alleles were present in 45.5% of NT H. influenzae but in less than 1% of H. haemolyticus strains (P < .0001). NT H. influenzae had an average of 26.8 tetranucleotide repeats in licA compared to 14.8 repeats in H. haemolyticus (P < .05). In addition, NT H. influenzae strains that possessed a licDIII allele had increased numbers of repeats compared to NT H. influenzae with other licD alleles (P < .05).These data demonstrate that genetic similarities and differences of ChoP expression exist between NT H. influenzae and H. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of NT H. influenzae.

Dataset Information

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

Publications

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

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