HYPERVARIABLE REGION-IV OF SALMONELLA GENE FLIC(D) ENCODES A DOMINANTSURFACE EPITOPE AND A STABILIZING FACTOR FOR FUNCTIONAL FLAGELLA

To identify the major antigenic determinant of native Salmonella flage lla of antigenic type d, we constructed a series of mutated flic(d) ge nes with deletions and amino acid alterations in hypervariable region IV and in regions of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T. M. Joys and F. Schodel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the muta nt genes, with deletions of up to 92 amino acids in region IV, assembl ed into functional flagella and conferred motility on flagellin-defici ent hosts. Serological analysis of these flagella with different anti- d antibodies revealed that the peptide sequence centered at amino acid s 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their banking sequence by a tripeptide specified by a li nker sequence eliminated most reactivity with antisera against wild-ty pe d flagella as tested by enzyme-linked immunosorbent assay or by Wes tern immunoblot. Functional analysis of the mutated flagellin genes wi th or without an insert suggested that amino acids 180 to 214 in the 5 ' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins fo rmed by insertion of peptide sequence pre-S1 12-47 of hepatitis B viru s surface antigen into the deleted flagellins assembled into functiona l flagella, and antibody to the pre-S1 sequence was detected after imm unization of mice with the hybrid protein. This suggests that such mut ant flagellins containing heterologous epitopes have potential as vacc ines.