Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (Oncorhynchus mykiss) skin and gill

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.

An Overview of the Immunological Defenses in Fish Skin

“…Despite the intensive research on AMPs in animals, there is still surprisingly little data documenting their in vivo antimicrobial upregulation [185,186]. Low levels of antimicrobial peptides from healthy specimens or other factors such as pH could be the reason for difficulty in isolating antimicrobial peptides/polypeptides from mucus [121].…”

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

“…For example, although histone core particles are generally located within the cell nucleus as part of condensed chromatin, recent studies have isolated cytoplasmic and secreted histone proteins with antimicrobial activity from a wide range of organisms and tissues, such as mollusks [3], fish [4], amphibians [5], and the human gastrointestinal tract [6]. The antimicrobial role of histones has been discussed further in recent reviews [7, 8].…”

Evaluation of antibacterial activity and innate immune components in skin mucus of Indian major carp,Cirrhinus mrigala

“…A full-length antimicrobial histone H2A was isolated from the skin mucus of O. mykiss (Fernandes et al 2002). Histonelike antimicrobial proteins have been isolated from the skin of Ictalurus punctatus (Robinette, Wada, Arroll, Levy, Miller & Noga 1998), O. mykiss and hybrid Morone saxatilis male 9 M. chrysops female (Noga, Borron, Hinshaw, Gordon, Gordon & Seo 2011). Histone H2B with potent bactericidal activity was isolated from the skin mucus of G. morhua along with the other antimicrobial ribosomal proteins (Bergsson et al 2005).…”