Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (Oncorhynchus mykiss) skin and gill
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Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 μg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.
KeywordsAntimicrobial polypeptides Histones ELISA Immunoassay
We thank D. Butkus for assistance with the ELISAs; K. Stone, T. Abbott, and W. McMurray (Yale University) for assistance with sequencing; J. Johansen for assistance with western blotting; and S. Salger for assistance with statistical analysis. This study was supported by National Science Foundation Small Business Innovation Research project 0320324 and Yale School of Medicine’s Clinical and Translational Science (CTSA) Award UL1 R024139, National Center for Research Resources, National Institutes of Health. All research was approved and performed under North Carolina State University IACUC protocol #05-099-0.
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