Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.
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.
Lee
K K
KK
MRC Group in Protein Structure and Function, University of Alberta, Edmonton, Canada.
Sastry
P A
PA
Paranchych
W
W
Hodges
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D001666:Binding Sites, Antibody; D003429:Cross Reactions; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000032:analysis* / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000032:analysis* / Q000276:immunology; D011817:Rabbits
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D004220:Disulfides; D010446:Peptide Fragments; D036781:Fimbriae Proteins