STRUCTURE-FUNCTION ANALYSIS OF THE ADHERENCE-BINDING DOMAIN ON THE PILIN OF PSEUDOMONAS-AERUGINOSA STRAINS PAK AND KB7

The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue s ynthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal s equence of the PAK pilin protein (residues 128-144) contains the adher ence binding domain. Another pilin strain, KB7, has been cloned and se quenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, P athogenesis and Evolving Biotechnology, pp 343-351, American Society f or Microbiology, Washington, DC]. The C-terminal l7-residue sequence o f the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compar ed to the PAK sequence. In this study, the interactions between the A5 49 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-t erminal l7-residue synthetic peptide of the pilins. The ability of the se peptide analogs to inhibit the binding of the biotinylated PAK pill to A549 cells was assessed, Six PAK amino acid side chains (Ser(131), Gln(136), Ile(138); pro(139), Gly(141) ,and Lys(144)) and nine KB7 si de chains (Ala(130), Thr(131), Thr(132), Val(133), Asp(134), Ala(135), Lys(136), Arg(138), and Pro(139)) were found to be important in media ting the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pill to A549 cells. This suggests that t he interactions between the pilin ligand and the A549 cell surface rec eptors are dependent on the conformation mediated by the disulfide bri dge (Cys(129) and Cys(142)). The residues considered to contribute to bacterial adherence are referred to as the ''adhesintope''. Four PAK a nd three KB7 side chains were located in a structurally more rigid reg ion of the disulfide-bridged peptide as revealed by two-dimensional NM R studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The st ructural aspects of the pilin-receptor interactions related to the map ped adhesintope sequences are discussed. The dissimilarities between t he PAK and KB7 adhesintopes may suggest that compensatory mutations co uld occur among different pilin strains so as to allow the pilin adhes ins to interact with the same receptor.