|Wiens, Gregory - Greg|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/21/2004
Publication Date: 6/1/2005
Citation: Wiens, G.D., Owens, J. 2005. Mapping neutralizing epitopes on renibacterium salmoninarum p57 using transposon mutagenesis and synthetic peptides. Applied and Environmental Microbiology 71 (6): 2894-2901. Interpretive Summary: Renibacterium salmoninarum produces a 57-kDa protein (p57) that has been associated with the capacity to cause disease and is a reliable diagnostic marker of R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. Here, we map regions on p57 where neutralizing monoclonal antibodies bind. These antibodies bind to spatially separate regions on the amino-terminus of the protein as determined by transposon mutagenesis. Furthermore, recognition of secondary structure on the amino-terminus of p57 appears to be important for neutralization. The results from the epitope mapping studies suggest directions for improvement of antibody-based immunoassays for the detection of R. salmoninarum infected fish.
Technical Abstract: Renibacterium salmoninarum is a Gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and over-expressed in E. coli BL21 DE3. The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino-terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum infected fish.