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

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 ␣-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.

Antimicrobial egg cleaning by the fringed darter (Perciformes: Percidae:Etheostoma crossopterum): implications of a novel component of parental care in fishes

“…Nevertheless, the effect can still be viewed as antifungal given that the presence of the guarding male decreased in-stream fungal growth on eggs and the addition of mucus apparently eliminated fungal growth on eggs in the centrifuge tubes. The recent discovery of antimicrobial compounds in fish epidermal mucus has revealed a striking line of defence in response to the microbial composition of the aquatic environment (Austin & McIntosh 1988;Cole et al 1997;LeMaitre et al 1997;Ebran et al 2000). If adult fishes benefit from this defence, eggs deposited on the biofilm-covered substrate will also presumably benefit from a defence against microbial pathogens.…”

“…Previous studies of vertebrate antimicrobial compounds indicate that these substances are not specific, with inhibitory effects demonstrated on a broad range of microbes (Zasloff 1987(Zasloff , 2002Boman 1995;Cole et al 1997;LeMaitre et al 1997;Silphaduang & Noga 2001). Because a limited amount of mucus could be acquired from each E. crossopterum male, and relatively small population sizes in each stream limited the number of males that could be captured, we directed antimicrobial assays at bacterial and fungal strains that represented common characteristics of the microbial community.…”

“…Based on previous studies (Cole et al 1997;LeMaitre et al 1997), the epidermal mucus of fishes appears to serve as a medium for the concentration and, hypothetically, delivery of an antimicrobial agent. We used three separate samples of E. crossopterum mucus to determine the effect of mucus on bacterial growth.…”

“…Oncorhynchus mykiss (Austin & McIntosh 1988); Cyprinus carpio (Cole et al 1997); Pleuronectes americanus (LeMaitre et al 1997); Morone saxatilis´M. chrysops hybrid (Silphaduang & Noga 2001)).…”

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

“…Over 800 AMP sequences have been deposited in the Antimicrobial Peptide Database (http://bbcm1.univ.trieste.it/∼tossi/pag1.htm). Increasing numbers of antimicrobial peptides have been identified from teleost fishes in recent years (e.g., Cole et al, 1997;Douglas et al, 2001Douglas et al, , 2003aLauth et al, 2002;Shike et al, 2002Shike et al, , 2004Noga and Silphaduang, 2003;Zhang et al, 2004;Bao et al, 2005, in press;Xu et al, 2005). As the largest vertebrate group containing over 23,000 species (Helfman et al, 1997), teleosts should be a rich source of antimicrobial peptides.…”