Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.
Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.
D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000276:immunology / Q000648:ultrastructure*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D010455:Peptides / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D008565:Membrane Proteins; D010455:Peptides; D036781:Fimbriae Proteins