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Applied and Environmental Microbiology, June 2005, p. 2894-2901, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2894-2901.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Gregory D. Wiens1* and Jennifer Owen2

USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd., Kearneysville, West Virginia 25430,1 Department of Molecular Microbiology and Immunology, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, Oregon 972012

Received 2 November 2004/ Accepted 21 December 2004

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 overexpressed in Escherichia 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.

* Corresponding author. Mailing address: USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd., Kearneysville, WV 25430. Phone: (304) 724-8340. Fax: (304) 725-0351. E-mail: gwiens{at}ncccwa.ars.usda.gov.

Applied and Environmental Microbiology, June 2005, p. 2894-2901, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2894-2901.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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