|Wiens, Gregory - Greg|
Submitted to: Annual Eastern Fish Health Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 4/21/2003
Publication Date: 4/21/2003
Citation: Wiens, G.D, Owen, J., Pascho, R., Winton, J.R. 2003. Mapping antibody epitopes on renibacterium salmoninarum p57 using transposon mutagenesis and synthethic peptides. 28th Annual Eastern Fish Health Workshop. p.70.
Technical Abstract: Renibacterium salmoninarum is a gram-positive bacterium that causes salmonid bacterial kidney disease. Renibacterium salmoninarum produces a large amount of a cell-surface and secreted 57-kDa protein (p57) that binds and agglutinates salmon leukocytes and rabbit red blood cells. The location of the p57 cell-binding domain is unknown. Previously, we identified three monoclonal antibodies (4D3, 4C11, and 4H8) that block the agglutinating activity of p57. These MAbs bind to a recombinant, amino-terminal fragment of p57 containing amino acids 32 through 243. Here, we map monoclonal and polyclonal antibody epitopes using transposon mutagenesis and synthetic peptides to characterize the functional domains and immunodominant regions of p57. A Tn5 based transposon was used to introduce a 19 amino acid insertion randomly into a truncated, p57 protein. Forty-four mutants were generated and over-expressed in E. coli BL21 DE3. The binding of MAbs 4D3, 4H8, and 4C11 to each mutant protein was determined by Western blotting. Transposons inserting between amino acids 51 and 111 disrupted the 4H8 epitope. Insertions between residues 51 and 209 disrupted the 4C11 epitope, and insertions between amino acids 158 and 233 disrupted the 4D3 epitope. To further map the MAb epitopes, 15-mer biotinylated peptides spanning p57 were synthesized and tested in a direct binding ELISA. MAbs 4D3, 4C11 and 4H8 failed to bind peptides spanning the amino-terminus p57, while two other MAbs (4D10 and 1A1) bound strongly to peptides from the carboxy-terminal region of p57. Many of the peptides were recognized by goat anti-R. salmoninarum antisera and chinook salmon anti-p57 antisera suggesting functional availability of the peptides for binding. Interestingly, the goat and chinook salmon polyclonal antisera exhibited distinct patterns of peptide recognition. Taken together, these data suggest that many peptides were functional and that the epitopes recognized by MAbs 4D3, 4C11 and 4H8 may be discontinuous in conformation. Our data are consistent with a model of the involvement of the amino-terminus of p57 in leukocyte binding and suggest that secondary structure is important for MAb antigen-recognition. Peptides spanning p57 may be a useful tool for further characterization of polyclonal antisera and the comparative analysis of immune recognition.