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Infect Immun. 1989 February; 57(2): 520-526

Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.

MRC Group in Protein Structure and Function, University of Alberta, Edmonton, Canada.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen that attaches to host cells via their pili. The pilus of P. aeruginosa PAK consists of a polymer of a single subunit, pilin, which is a 144-residue polypeptide. The C-terminal end of this protein is semiconserved in a number of strains and contains a disulfide bridge. We have synthesized the C-terminal peptide PAK (128-144)-OH in both its reduced and oxidized forms and the analog PAK(A-129) (128-144)-OH, in which cysteine-129 was substituted by alanine. These three peptides were used to immunize rabbits and prepare antipeptide antisera. It was found that antipeptide antisera to reduced peptide (17-R) and to oxidized peptide (17-O) bound to native PAK pili and cross-reacted with strain PAO pili in direct enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. However, the antiserum to the peptide immunogen PAK(A-129)(128-144)-OH, which does not have the ability to form the disulfide bridge, did not bind to either PAK or PAO pili. Competitive ELISA experiments with reduced and oxidized peptides of Ac-PAK(128-144)-OH showed that there was no difference in binding between the two peptides for 17-R or 17-O immunoglobulin G. When immunoglobulin G from native PAK antipilus antiserum was used in competitive or direct ELISA experiments, there was also no preference in binding to reduced or oxidized Ac-PAK(128-144)-OH or to PAK(A-129)(128-144)-OH. This result showed that the disulfide bridge in Pseudomonas pili is not critical to the immunogenicity of this region. However, the disulfide bridge is important in the immunogenicity of the C-terminal peptide when preparing antipeptide antisera that are cross-reactive with pili from different strains, since only the disulfide bridge peptide antisera cross-reacted well with the PAO pili as shown by competitive ELISA, suggesting that this region could be an important candidate for development of a synthetic vaccine.