Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) skin and gill |
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| Authors: | Edward J Noga Paul J Borron Jeffrey Hinshaw William C Gordon Linda J Gordon Jung-Kil Seo |
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| Institution: | (1) Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA;(2) Norcarex Bio Corporation, 3045 Granville Drive, Raleigh, NC 27609, USA;(3) Department of Biology, College of Agriculture and Life Sciences, North Carolina State University, 455 Research Drive, Mills River, NC 28759, USA;(4) Bethyl Laboratories, 25043 West FM 1097, Montgomery, TX 77356, USA;(5) Present address: RTI International, 3040 Cornwallis Road, PO Box 12194, Research Triangle Park, NC 27709-2194, USA;(6) Present address: Alpha Diagnostics International, Inc., 6203 Woodlake Center Drive, San Antonio, TX 78244, USA |
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| Abstract: | 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. |
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