Role of a 22-kilodalton pilin protein in binding of Pseudomonas cepacia to buccal epithelial cells.

Previously we have shown that many isolates of Pseudomonas cepacia obtained from the sputum of patients with cystic fibrosis exhibit specific binding to purified mucins. The binding was mediated by a 22-kDa protein located on peritrichous pili of the bacteria. Nonpiliated bacteria did not bind to mucin. In the present study we found that both piliated and nonpiliated P. cepacia bind to buccal epithelial cells (BECs) obtained from health human volunteers. Scatchard plot analyses of binding data with the LIGAND computer program suggest the presence of at least two classes of cell receptors (A and B) for piliated P. cepacia (isolates PC 5 and PC 7) and a single class of receptors (A) for nonpiliated P. cepacia (isolates PC 45 and PC 61). The affinity constants for receptor A varied from 1.7 x 10(-9) to 4.7 x 10(-8) ml/CFU. Receptor B had a lower affinity constant (2.5 x 10(-10) to 1.2 x 10(-9) ml/CFU) but a greater saturation capacity. Receptor B was similar in affinity to the mucin receptor for piliated P. cepacia (3.3 x 10(-10) to 1.3 x 10(-9) ml/CFU). Purified mucin partially inhibited the binding of piliated bacteria to BECs by competing with BEC receptor site B. The purified 22-kDa pilin adhesin and an antiadhesin antibody also caused partial inhibition. One BEC receptor for piliated isolates of P. cepacia was identified as a 55-kDa protein as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BEC homogenate supernatants, Western blotting (immunoblotting), and bacterial overlay assays. Preincubation of piliated bacteria with either mucin or the antiadhesin antibody abolished binding to the 55-kDa BEC receptor. In summary, our results indicate that piliated P. cepacia interacts with BECs by using at least two different adhesin-receptor systems. One adhesin (not examined) is common to piliated and nonpiliated P. cepacia, but the other system is the pilus-localized 22-kDa mucin-binding adhesin and its 55-kDa BEC receptor protein. Because it mediates adherence to both mucin and epithelial cells, the 22-kDa adhesin may be an important virulence determinant in cystic fibrosis lung infections in which mucins are abnormally adhesive on mucosal surfaces.