microbiology, Vol 142, 3261-3268, Copyright © 1996 by Society for General Microbiology

ARTICLES

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

P Everest, J Li, G Douce, I Charles, J De Azavedo, S Chatfield, G Dougan and M Roberts
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.

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.

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