Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder

Alexander M Cole,Peddrick Weis,Gill Diamond

doi:10.1074/jbc.272.18.12008

Alexander M Cole, Peddrick Weis + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.18.12008

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Abstract

Antimicrobial peptides are found in both myeloid cells and mucosal tissues of many vertebrates and invertebrates. These peptides are predicted to operate as a first-line host defense mechanism exerting broad-spectrum activity against pathogenic bacteria, fungi, parasites, and enveloped viruses. We report the characterization of a novel 25-residue linear antimicrobial peptide found in the skin mucous secretions of the winter flounder (Pleuronectes americanus). This peptide was purified through multiple chromatographic methods to obtain a single peak by reversed-phase high performance liquid chromatography. This purified peptide, which we named pleurocidin, exhibited antimicrobial activity against Escherichia coli in a bacterial cell lysis plate assay. Mass spectrometry and amino acid sequence analysis indicated that it is 25 amino acids in length. Pleurocidin is predicted to assume an amphipathic alpha-helical conformation similar to many other linear antimicrobial peptides. There is a high degree of homology between pleurocidin and two antimicrobial peptides, ceratotoxin from the Mediterranean fruit fly and dermaseptin from the skin of a hylid frog. The minimal inhibitory concentration and minimal bactericidal concentration of pleurocidin were determined against 11 different Gram-negative and Gram-positive bacteria. Immunohistochemistry locates pleurocidin in the epithelial mucous cells of flounder skin. Pleurocidin represents a novel antimicrobial peptide found in fish and may play a role in innate host defense.

Highlights

Antimicrobial peptides are among the earliest developed molecular effectors of innate immunity and are significant in the first line of the host defense response of diverse species [1]
Skin secretions of winter flounder were assayed for antimicrobial peptides based on methods designed for similar studies in amphibians [13]
These fractions correspond to the peptide region (Ͻ5000 Da) when analyzed by SDS-polyacrylamide gel electrophoresis. When these fractions were pooled and subjected to ion exchange HPLC, a single peak which eluted at 25.7 min (Fig. 1B) was determined to contain the antimicrobial activity. This was further purified by reversed-phase HPLC, which resulted in a single peak at 29.1 min (Fig. 1C) which exhibited antimicrobial activity

Summary

EXPERIMENTAL PROCEDURES

Isolation and Purification of Antimicrobial Peptides from Fish Skin—Winter flounder epidermis and mucus extracts, isolated by. A single 220-nm absorbing fraction eluting at 25.7 min, which possessed antimicrobial activity, was applied to a Vydac 218TP C18 (5-␮m particle size, 4.6 –250 mm) reversed-phase HPLC column (linear AB gradient where A is H2O 0.1% trifluoroacetic acid, and B is acetonitrile, 0.1% trifluoroacetic acid, with a 45-min gradient at 1.33% acetonitrile/min). The MIC was determined by incubating serial dilutions of synthetic pleurocidin with approximately 1 ϫ 103 bacterial colony-forming units in a 96-well microtest culture dish. Kinetic Analysis—1 ϫ 103 E. coli colony-forming units were incubated at 37 °C for increasing times (1 min to 8 h) with one of three concentrations of pleurocidin (0.5 ϫ, 1 ϫ, and 2 ϫ MIC). To determine optimal NaCl concentration for pleurocidin activity, a modified MIC protocol was performed with E. coli

Antimicrobial Peptide from Skin of Winter Flounder

RESULTS

DISCUSSION