BURKHOLDERIA (BASONYM PSEUDOMONAS) CEPACIA BINDING TO LIPID RECEPTORS

Piliated Burkholderia (formerly Pseudomonas) cepacia from sputa of cys tic fibrosis patients in Toronto, Canada, were shown earlier to bind t o purified mucins and to a protein receptor on epithelial cells via a 22-kDa adhesin located on unique cable pili. However, a second recepto r, thought to be lipid in nature, was also identified on cells and app eared to serve as the major cell receptor for poorly piliated or nonpi liated isolates. In the present study in vitro approaches were used to identify putative lipid receptors for B. cepacia and to explore the n ature of the binding interaction. As judged by thin-layer chromatograp hy overlay assays, the best receptors were digalactosylceramide and gl obotriosylceramide (Gb(3)). Both contain an unsubstituted terminal Gal alpha 1-4Gal sequence. B. cepacia also bound moderately to galactosyl ceramide, gangliotriosylceramide, and gangliotetraosylceramide. Bindin g to Gb(3) of a nonpiliated isolate was stronger than that of piliated isolates, suggesting that nonpiliated isolates may have greater acces s to lipid receptors or a greater concentration of an adhesin for Gb(3 ). Binding to glycolipids was not affected by tetramethylurea, a hydro phobic-bond-breaking agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the carbohyd rate epitope to the bacteria. Gb(3) was also the major receptor in lip id extracts of human erythrocytes, human buccal epithelial cells and H Ep-2 laryngeal epithelial cells. In a receptor-based enzyme-linked imm unosorbent assay, binding to Gb(3) within a phospholipid-cholesterol m ixture (a membrane-like environment) increased and then approached sat uration as a direct function of increasing bacterial concentration. Th e calculated value of K-a (3.06 x 10(-8) ml/CFU), the affinity constan t, was almost identical to the K-a calculated earlier for B. cepacia b inding to a set of lipid receptors in buccal epithelial cells (1.5 x 1 0(-8) to 2.0 x 10(-8) ml/ CFU). Our findings suggest that,within cell membranes, galactose-containing glycolipids, particularly Gb(3), are g ood candidates for receptors for B. cepacia, particularly for isolates in which cable pill are poorly expressed.