THE PILI OF PSEUDOMONAS-AERUGINOSA STRAINS PAK AND PAO BIND SPECIFICALLY TO THE CARBOHYDRATE SEQUENCE BETA-GALNAC(1-4)BETA-GAL FOUND IN GLYCOSPHINGOLIPIDS ASIALO-GM(1) AND ASIALO-GM(2)

Pseudomonas aeruginosa employs pill to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM(1) (Lee ef at, 199 4 - accompanying article). PAK and PAO pill were examined for their ab ilities to bind to the synthetic beta GalNAc(1-4)beta Gal (a minimal s tructural carbohydrate receptor sequence of asialo-GM(1) and asialo-GM (2) proposed by Krivan at al., 1988a) using solid-phase binding assays . Both pill specifically bound to beta GalNAc(1-4)beta Gal. The bindin g of beta GalNAc(1-4)beta Gal-Biotin to the immobilized PAK and PAO pi ll was inhibited by corresponding free pill. The receptor binding doma in of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128-144) of the pilin structural subunit (Irvin at al., 198 9). Biotinylated synthetic-peptides corresponding the C-terminal resid ues 128-144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the beta GalNAc(1-4)beta Gal-(bovine serum albumin (BSA)). Th e binding of biotinylated peptides to beta GalNAc(1-4)beta Gal-BSA was inhibited by PAK pill, Ac-KCTSDQDEQFIPKGCSK-OH (AcPAK(128-144)(ox)-OH ) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128-144)(ox)-OH) peptides. (In th ese peptides Ac denotes Na-acetylation of the N-terminus, -OH means a peptide with a free alpha-carboxyl group at the C-terminus and the 'ox ' denotes the oxidation of the sulphhydryl groups of Cys-129 and Cys-1 42.) Both acetylated peptides were also able to inhibit the binding of beta GalNAc(1-4)beta Gal-biotin to the corresponding BSA-Peptide(128- 144)(ox)-OH conjugates. The beta GlcNAc(1-3)beta Gal(1-4)beta Glc-biot in conjugate was unable to specifically bind to either immobilized PAK and PAO pili or the respective C-terminal peptides. The data above de monstrated that the P. aeruginosa pill recognize asialo-GM(1) receptor analogue and that beta GalNAc(1-4)beta Gal disaccharide is sufficient for binding. Furthermore, the binding to beta GalNAc(1-4)beta Gal was mediated by residues 128-144 of the pilin subunit.