title: Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells.
abstract: The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4 degrees C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD-->RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Everest
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Douce
authors.forename: I
authors.initials: I
authors.lastname: Charles
authors.forename: J
authors.initials: J
authors.lastname: De Azavedo
authors.forename: S
authors.initials: S
authors.lastname: Chatfield
authors.forename: G
authors.initials: G
authors.lastname: Dougan
authors.forename: M
authors.initials: M
authors.lastname: Roberts
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology*; D001886:Bordetella pertussis / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity; D005798:Genes, Bacterial; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010957:Plasmids / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics / Q000472:pathogenicity; D014774:Virulence; D010566:Virulence Factors, Bordetella
chemical_list: D001425:Bacterial Outer Membrane Proteins; D009842:Oligopeptides; D010566:Virulence Factors, Bordetella; C068929:pertactin; C047981:arginyl-glycyl-aspartic acid