Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide ...

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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 272, No. 18, Issue of May 2, pp. 12008 –12013, 1997 Printed in U.S.A.

Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder* (Received for publication, December 20, 1996, and in revised form, February 25, 1997)

Alexander M. Cole, Peddrick Weis, and Gill Diamond‡ From the Department of Anatomy, Cell Biology and Injury Sciences, UMDNJ-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103

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 a-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 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). Many different families of molecules have been found throughout the animal and plant kingdoms that display similar modes of action against a wide range of microbes (1). Each family of peptides shares several common properties. They tend to display broad-spectrum antimicrobial activity and cationic charge at physiological pH. Many of these peptide families are expressed in more than one cell type and in more than one species (1). In addition to microbicidal capabilities, certain peptides also confer diverse functions such as promotion of wound healing (2) and stimulation of monocyte chemotaxis (3). * This research was supported in part by the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce (Grant NA36RG050, Project R/N-95003). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This report is New Jersey Sea Grant Publication No. NJSG-96-394. ‡ To whom correspondence should be addressed: University of Medicine and Dentistry of New Jersey, Dept. of Anatomy, Cell Biology and Injury Sciences, 185 South Orange Ave., Newark, NJ 07103. Tel.: 201982-3324; Fax: 201-982-7489; E-mail: [email protected].

While research has shown that vast quantities of antimicrobial peptides are found in inflammatory cells of most species studied (reviewed in Refs. 4 – 6), recent interest has been directed toward the mucosal epithelia (7–10) (as reviewed in Ref. 11). The mucosal epithelial layer of many species acts as a physical barrier to the harsh external environment. Consequently most species tested have been found to contain antimicrobial agents at these sites. Antimicrobial peptides in the mucosal tissue include andropin, a reproductive tract epithelial peptide from Drosophila (12); magainin, from granular glands of Xenopus laevis (9, 13); dermaseptin, from the skin of the arboreal frog Phyllomedusa bicolor (14); tracheal antimicrobial peptide, from the columnar epithelial cells of the bovine trachea (7); and enteric defensins in the mammalian gastrointestinal tract (10, 15). Frog skin produces a number of biologically active peptides including magainin (16) and dermaseptin (14, 17). These linear peptides were shown to be antibacterial, antifungal, and antiprotozoal (17, 18). Both types of antimicrobial peptides have been shown to adopt an amphipathic a-helical conformation in hydrophobic media (14, 19, 20). It has been suggested that this structural type of peptide binds anionic phospholipid-rich membranes similar to bacterial membranes and dissolves them like detergents (21–23). Natural antibiotics, which are not structurally homologous to magainin, dermaseptin, or ceratotoxin, have been isolated from several aquatic species. Pardaxin, a 33-amino acid poreforming polypeptide toxin originally deemed a shark repellent peptide from sole (reviewed in Shai (24)), has recently been determined to exert antibacterial activity (25). This peptide possesses a helix-hinge-helix structure and, unlike amphipathic a-helical peptides, is also cytotoxic to mammalian cells. Most recently two proteins (27 and 31 kDa) have been isolated which confer antibacterial properties in the skin mucus of carp (26). While these proteins have been discovered to induce ion channels in planar lipid bilayers, their sequence, structure, and function have not been determined. Squalamine, a cationic steroidal antibiotic isolated from the dogfish shark, has been shown to exert broad-spectrum antimicrobial action against Gram-negative and -positive bacteria as well as fungi and protozoa (27, 28). Many marine species possess a mucosal barrier to the microbe-laden external environment and therefore should possess an innate host defense mechanism to combat infection. We therefore examined the skin secretions of winter flounder (Pleuronectes americanus) for the existence of antimicrobial peptides. We report the discovery and characterization of a novel 25-residue antimicrobial peptide, pleurocidin, that is produced in the epidermal mucous cells of winter flounder. EXPERIMENTAL PROCEDURES

Isolation and Purification of Antimicrobial Peptides from Fish Skin—Winter flounder epidermis and mucus extracts, isolated by

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Antimicrobial Peptide from Skin of Winter Flounder scraping, were homogenized in a solution of 50 ml of 0.2 M sodium acetate, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 20 min at 20,000 3 g (Beckman JA-17 rotor), applied to SepPak Vac 1g C18 cartridges (surface pH 7.0, 12% carbon, 12.5-nm pore size, 80-mm particle size) for solid phase extraction, and eluted with 60% acetonitrile, 0.1% trifluoroacetic acid. The dried eluate was resuspended in 50 mM Tris-HCl and subjected to size fractionation by Sephadex G-50 chromatography in 50 mM ammonium formate, pH 5.1, with absorbance monitoring at 215 nm. Fractions were lyophilized, resuspended in a small volume of water, and assayed for antimicrobial activity using a standard bacterial lysis plate assay (Escherichia coli on LB medium agarose plate supplemented with 50 mM NaF) as described previously (7). Fractions displaying a microbicidal zone of clearing were pooled and subjected to strong Poly-LC polysulfoethyl (5-mm particle size, 4.6 3 200 mm) cation-exchange HPLC1 (linear AB gradients where A is 25% acetonitrile, 5 mM KH2PO4, pH 5.0, and B is 25% acetonitrile, 5 mM KH2PO4, pH 5.0, 1 M NaCl, with a 45-min gradient at 22.2 mM NaCl/min). A single 220-nm absorbing fraction eluting at 25.7 min, which possessed antimicrobial activity, was applied to a Vydac 218TP C18 (5-mm 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). A single peak eluting at 29.1 min was determined to contain the antimicrobial activity. Protein Characterization—Heat stability was tested by boiling for 5 min. Verification of peptide nature was performed by exposure to proteinase K for 30 min at 37 °C. Matrix-assisted laser desorption time-offlight mass spectrometry, amino acid analysis, and peptide sequence analysis were performed by the Harvard Microchemistry Facility (Cambridge, MA). Computer analysis of the peptide was carried out using MacVector software (IBI-Kodak) and GCG (29). Peptide Synthesis and Antibody Production—Pleurocidin was synthesized using solid phase technology, quantitated by HPLC analysis, and conjugated with a keyhole limpet hemocyanin carrier to obtain polyclonal rabbit antisera (Research Genetics, Huntsville, AL). Monospecific polyclonal antibodies to pleurocidin were affinity-purified using an AminoLink Plus Immobilization Kit (Pierce). Briefly, 1 mg of pleurocidin was introduced to a solid phase matrix (4% cross-linked beaded agarose) support for coupling through primary amines. The resultant covalent linkage immobilized the antigen to the support. Bacteria and Culture Conditions—E. coli, strain D31, was cultured in LB, 37 °C; Leucothrix mucor from eggs of winter flounder (ATCC 25907) was cultured in OZR medium, 26 °C; Aeromonas salmonicida subsp. salmonicida from salmon skin (ATCC 49385) was cultured in Trypticase Soy broth/agar, 26 °C; Cytophaga aquatilis from gills of diseased salmon (ATCC 29551) was cultured in nutrient broth/agar, 22 °C; and Pasteurella hemolytica from bovine respiratory tract was cultured in brain heart infusion medium, 37 °C. Serratia marcescens, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium I, and S. typhimurium II were cultured in trypticase soy broth/agar, 37 °C. Bacteriostatic and Bactericidal Analysis—The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were obtained to determine bacteriostatic and bactericidal activities, respectively, for each strain of bacteria listed above. The MIC was determined by incubating serial dilutions of synthetic pleurocidin with approximately 1 3 103 bacterial colony-forming units in a 96-well microtest culture dish. The lowest concentration which inhibited bacterial growth was deemed the MIC. The MBC was determined by spreading 5 ml from each well of the MIC on an LB agar plate and incubating overnight. The MBC was indicated by the concentration of pleurocidin that inhibited growth. Note that, although agar-based assays of antimicrobial activity have been shown to underestimate the antibacterial activity of Gram-negative microorganisms when compared with liquid-based assays (30), the agar-based assays used in this study quantified remaining viable colonies and not antimicrobial activity. Kinetic Analysis—1 3 103 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 3, 1 3, and 2 3 MIC). The reactions were terminated by plating on LB agar, and plates were incubated overnight at 37 °C. To determine optimal NaCl concentration for pleurocidin activity, a modified MIC protocol was performed with E. coli

1 The abbreviations used are: HPLC, high performance liquid chromatography; MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration.

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FIG. 1. Purification of an antimicrobial peptide from fish skin. A, absorbance at 215 nm for each fraction eluted off a Sephadex G-50 size fractionation column. Antimicrobial section represented by bracket. B, ion-exchange HPLC chromatogram. Peak fraction indicated by the arrow (;25.7 min) was subjected to final reversed-phase HPLC. Dotted line indicates the NaCl concentration. C, final reversed-phase HPLC. Peak fraction eluting at 29.1 min was shown to be antimicrobial. Dotted line indicates the acetonitrile concentration.

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Antimicrobial Peptide from Skin of Winter Flounder

FIG. 2. Primary amino acid sequence of pleurocidin and homology with other antimicrobial peptides. Homologous residues in several members of dermaseptin and ceratotoxin classes of antimicrobial peptides are shown in italicized bold. Numbers in parentheses to the right of each sequence indicate a ratio of homologous residues to total number of residues in the conserved region (20-residue area forming the putative a-helix). Scores, in percent, are also listed. using LB without NaCl (10 g of tryptone, 5 g of yeast extract) and adding NaCl to cultures at increasing concentrations. 1 3 103 E. coli colony-forming units were initially incubated for 4 h at 37 °C with varying concentrations of pleurocidin (0 –5.6 mM) and NaCl (0 –1.25 M). Reactions were plated on LB agar plates and incubated overnight at 37 °C. Immunohistochemistry—Fresh skin sections were obtained, immediately fixed in 4% buffered paraformaldehyde for 4 h, dehydrated, and embedded in paraffin. Serial cross sections (5 mm) of flounder skin were deparaffinized in xylene and rehydrated. Briefly, slides were blocked with goat serum (20 min) and incubated at varied concentrations of either preimmune serum, primary rabbit polyclonal antiserum, or monospecific affinity-purified polyclonal antibody diluted in goat serum overnight at 4 °C. After washing with phosphate-buffered saline, slides were incubated with biotinylated goat anti-rabbit antibody, followed by streptavidin-conjugated alkaline phosphatase. Enzyme activity was detected with nitro blue tetrazolium/4-bromo-5-chloro-3-iodolylphosphate solution as dye substrates for alkaline phosphatase. The desired signal level was achieved after 18 min of incubation. Slides were then counterstained for 5 min in Light Green (Sigma). Photography was carried out with a Zeiss photomicroscope using Kodak ASA100 film. RESULTS

Skin secretions of winter flounder were assayed for antimicrobial peptides based on methods designed for similar studies in amphibians (13). Secretions from ten fish, collected by scraping, were extracted in buffer. This extract was concentrated and subjected to gel filtration. Fig. 1A reveals a UV absorbance profile after Sephadex G-50 gel chromatography and indicates antimicrobial activity in fractions 37– 47 (under bracket). These fractions correspond to the peptide region (,5000 Da) when analyzed by SDS-polyacrylamide gel electrophoresis (data not shown). 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. Notably, on both the ion exchange and reversed-phase HPLC columns, this antimicrobial peptide had retention times which are very similar to those of magainin and defensin. Antimicrobial activity was retained even when the fraction was subjected to boiling for 5 min. However, treatment with 20 mg/ml proteinase K for 30 min at 37 °C indicates that all antimicrobial activity is abolished after proteolytic digestion (data not shown). Structure and Sequence Characterization of Fish Skin Antimicrobial Peptide—The microbicidal reversed-phase HPLC fraction, resuspended in water, was subjected to mass spectral analysis, which determined a single ion cluster at m/z 2711. After completion of 23 rounds of N-terminal Edman degradation sequence analysis, two amino acids still remained to be sequenced. From combined amino acid HPLC analysis and mass spectrometry it was found that the two C-terminal residues were a tyrosine and leucine. Collisionally activated dissociation on a Finningan TSQ700 triple quadrupole mass spectrometer, followed by Edman chemistry and amino acid analysis, confirmed the final order of these last two amino acids

(Harvard Microchemistry Facility). The mature peptide sequence is given in Fig. 2. Since this peptide originates from the genus Pleuronectes and is determined to be bactericidal, we named the peptide pleurocidin. Searches against protein data bases indicated significant sequence identity to pleurocidin with the dermaseptin and ceratotoxin classes of antimicrobial peptides, which suggests homology. Sequence alignments are shown in Fig. 2. The dermaseptins and ceratotoxins have been proposed to form amphipathic a-helices (14, 31). Schiffer-Edmundson helical wheel modeling was employed to predict hydrophobic and hydrophilic regions within the secondary structure of pleurocidin (32). Fig. 3 depicts pleurocidin in an amphipathic a-helical conformation, indicating hydrophobic and hydrophilic residues on opposing sides of a 20amino acid central segment of the pleurocidin sequence. Also note that the hydrophilic surface is cationic in nature. Bactericidal Activity of Pleurocidin—Pleurocidin was tested against 11 Gram-positive and Gram-negative bacteria (Table I) for bactericidal and bacteriostatic activity. Bacteria were chosen which represent both mucosal and general pathogens. Three fish-host bacteria were used: A. salmonicida (isolated from skin of salmon), C. aquatilis (isolated from gills of diseased salmon), and L. mucor (from eggs of winter flounder). Results show that this peptide is active against both Gramnegative and Gram-positive bacteria. Most notably, we see that E. coli and B. subtilis are most sensitive to pleurocidin while L. mucor, S. marcescens, and P. aeruginosa are most resistant. In general, the bacteriostatic concentration (MIC) of pleurocidin is equivalent to the bactericidal concentration (MBC) of this peptide (except for A. salmonicida, which possesses a slightly higher bactericidal resistance). To determine the rate of bactericidal activity of pleurocidin, a kinetic study was performed using E. coli (Fig. 4). We demonstrate a time-dependent mechanism of action with increased bacteria-pleurocidin incubation leading to higher microbicidal activity. At 2 3 MIC (7.0 mM), antibacterial action increased dramatically after 15 min, and at 30 min only a few colonyforming units remained. At 1 3 MIC (3.5 mM), antibacterial action was noted after 30 min with few colony-forming units at 120 min. At 0.5 3 MIC (1.7 mM), antimicrobial action started after 120 min of incubation and most of the bacteria were not killed unless incubated with pleurocidin for 480 min. Also note that the grand mean, indicating average percent of control colony-forming units, increases with decreasing pleurocidin concentration. Winter flounder seasonally migrate from salt concentrations of approximately 0.8% in winter brackish waters to 3.3% in sea water. To determine whether the microbicidal action of pleurocidin is dependent on salt, NaCl concentration was varied with fixed concentrations of pleurocidin (Fig. 5) and allowed to proceed in bacteriostatic and bactericidal reactions as described for Fig. 4. We determined that pleurocidin is salt-

Antimicrobial Peptide from Skin of Winter Flounder

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FIG. 3. Schiffer-Edmundson helical wheel diagram demonstrating probable amphipathic a-helical conformation of pleurocidin. Boxes indicate hydrophobic amino acids. Residue numbers starting from amino terminus are shown. TABLE I Bacteriostatic and bactericidal activity of pleurocidin against 11 different fish-, sheep-, and human-specific pathogens Strain (Gram 1/2)

MIC

MBC

mM

Escherichia coli (2) Aeromonas salmonicida (2) Cytophaga aquatilis (2) Leucothrix mucor (1) Pasteurella haemolytica (2) Serratia marcescens (2) Bacillus subtilis (1) Pseudomonas aeruginosa (2) Staphylococcus aureus (1) Salmonella typhimurium I (2) S. typhimurium II (2)

2.2–3.3 17.7–35.0 2.2–4.4 .35.0 4.4–8.8 .35.0 1.1–2.2 .35.0 17.7–35.0 8.8–17.7 8.8–17.7

2.2–4.4 .17.7–35.0 2.2–4.4 .35.0 4.4–8.8 .35.0 1.1–2.2 .35.0 17.7–35.0 8.8–17.7 8.8–17.7

insensitive at physiological salt concentrations. Furthermore, pleurocidin can actively kill bacteria up to 625 mM NaCl, the approximate concentration of salt in sea water. Pleurocidin Is Found in Mucous Cells of Flounder Skin— Immunohistochemical methods were performed to determine the histological location of pleurocidin within a cross section of flounder skin. We determined that pleurocidin is concentrated in the epithelial mucous cells by using polyclonal antisera (Fig. 6A) and affinity-purified antibody (Fig. 6B), with preimmune sera as a control (Fig. 6C). The dark purple stain in these glandular cells is indicative of stored pleurocidin in mucincontaining packets. This is in contrast to the absence of stain in the mucous cells of the control slide. Some nonspecific staining of other epithelial cells and submucosa is evident in both the control and test sera as well as in the affinity-purified sample. When blocking incubation times were subsequently increased, little to no reduction in background was apparent. DISCUSSION

We report here the discovery of pleurocidin, a 25-residue peptide with broad-spectrum antimicrobial activity, in the skin secretions of the winter flounder. Pleurocidin retains sequence homology with the dermaseptin and ceratotoxin classes of an-

FIG. 4. Kinetics of pleurocidin-bacteria activity versus E. coli. Viable colonies shown as percent of control (without pleurocidin, plated at time 0). A, 7.0 mM (2 3 MIC); B, 3.5 mM (1 3 MIC); C, 1.7 mM (0.5 3 MIC). Dotted horizontal line indicates the grand mean.

timicrobial peptides (14, 17, 33). It is predicted to form an amphipathic a-helical structure similar to many other antimicrobial peptides, which exert their function by forming holes in the bacterial membrane (14, 19, 20, 34 –36). Pleurocidin exerts microbicidal action against a wide range of both Gram-negative and Gram-positive bacteria from various aquatic species implying broad-spectrum antibacterial action. This study indicates that fish, like several other aquatic species, possess in-

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FIG. 5. Microbicidal activity of pleurocidin versus E. coli is salt-independent. 100% killing determined as no bacterial growth after 18 h of incubation on agar plates; 0% killing indicates $1000 colony-forming units after 18 h of incubation. Dotted line represents the initial 4-h incubation with 5.6 mM pleurocidin; solid line represents incubation without pleurocidin.

nate host defense mechanisms to combat microbes on their mucosal surfaces. Interestingly, L. mucor, a bacterial species indigenous to the surface of winter flounder eggs, is resistant to pleurocidin’s action. This phenomenon suggests a bacterial mechanism that has evolved to counter the attack of antimicrobial peptides. Antimicrobial peptides are known to exert action by binding to the surface of microbial membranes and causing a lysis of the intracellular contents. Magainin, for example, exists in aqueous solution with a random secondary structure (37). It is the binding of magainin to an anionic-rich phospholipid surface that allows the peptide to assume an amphipathic a-helical conformation, with hydrophobic and hydrophilic sides segregating themselves. Both dermaseptins (14, 17, 38) and ceratotoxins (31, 33) are hypothesized to form an amphipathic a-helix in the region that could purportedly span a bacterial membrane, approximately 20 –21 amino acids in helical length. Since the primary sequences of pleurocidin, dermaseptin, and ceratotoxin are highly conserved, it is reasoned that their structure will also be conserved when these peptides are found to interact with the bacterial membrane. Amphipathic a-helical peptides, such as dermaseptin, ceratotoxin, and magainin, have been suggested to bind anionic phospholipid-rich membranes similar to bacterial membranes, and dissolve them like detergents (21–23). This mode of action may be characteristic of pleurocidin. Our kinetic study indicates a direct relation between concentration of peptide, incubation time, and bactericidal activity. This result has been demonstrated with other antimicrobial peptides. One aspect of this study involved varying the concentration of NaCl to determine pleurocidin’s salt dependence. As winter flounder migrate, depending on the season, their natural habitat can vary from brackish water (;0.8% salinity) to sea water (;3.3% salinity). It was determined that salt concentration did not have an effect on pleurocidin’s antibacterial action. Furthermore, NaCl only partially inhibited the microbicidal capability of pleurocidin at the high concentrations that approximate sea water salinity (3.3% > 565 mM). At extremely high salt concentrations, bactericidal activity was almost exclusively a direct effect of NaCl (Fig. 5). Smith et al. (39) recently discovered that the high NaCl concentration in the pulmonary mucosa of cystic fibrosis pa-

FIG. 6. Immunohistochemistry of cross-sectioned flounder skin. A, primary antisera, 1:5000 dilution in goat serum; B, affinitypurified monospecific polyclonal antibody, 1:1 dilution in goat serum; C, control, preimmune slide, 1:5000 dilution in goat serum. Arrows indicate epithelial mucous cells. Bar 5 50 mm.

tients leads to a decreased ability to kill bacteria, and inevitably aids in the pathogenesis of lung disease. Since pleurocidin exhibits bactericidal action at high (i.e. well above physiological) NaCl concentrations, the introduction of this salt-resistant antimicrobial peptide may prove beneficial in developing more effective antibiotic therapies for cystic fibrosis. The structure and function of pleurocidin differs greatly from other classes of antimicrobial molecules in marine species. Pleurocidin is located in the general epithelial mucous cells of the flounder, while pardaxin is found only in specific mucous glands that line the dorsal and anal fins of sole (40). Carp antibacterial proteins are roughly ten-fold larger (27–31 kDa) than pleurocidin (2.7 kDa), and the 27-kDa protein is glycosylated (26). Both proteins purportedly form large ion channels in the bacterial membrane in a manner similar to insect defensins. Furthermore, it has been shown that larger antibacterial proteins, such as aplysianin A from the sea hare (41) and achacin from the giant African snail (42), are constitutively secreted rather than secreted under stimulation (i.e. injury). Immunohistochemical data from our studies indicate a large concentration of pleurocidin resident within the mucous cells, thus suggesting that this antimicrobial peptide is stored until a suitable stimulation triggers release from the cells. This result is consistent with magainin localized to the granular glands in

Antimicrobial Peptide from Skin of Winter Flounder the skin of Xenopus and secreted upon activation (43). Histological analysis indicates that pleurocidin is localized in the mucous cells of the flounder skin. However, there may be additional explanations in what is noticed through immunohistochemistry. First, the positive staining in Fig. 6, A and B, shows a “spray” effect, indicative of release of mucous cell contents. It has yet to be determined whether this is mass secretion of the antimicrobial peptide or an artifact of tissue manipulation and preservation. Second, certain sections contained a dark purple stain at the epithelial surface of skin. This phenomenon may be attributable to microridges found on the surface of the skin which have been suggested to provide defense and assist in trapping mucous secretions, including pleurocidin, to the skin surface (44). In summary, we have isolated and characterized a novel 25-residue antimicrobial peptide, pleurocidin, from the skin of winter flounder with the amino acid sequence GWGSFFKKAAHVGKHVGKAALTHYL. This peptide has been shown to exert broad spectrum activity against a wide range of Grampositive and Gram-negative bacteria. Pleurocidin has been localized to the epithelial mucous cells of the flounder skin. It has high amino acid sequence homology with the dermaseptin and ceratotoxin classes of antimicrobial peptides. This homology, which is retained across widely diverse species, lends credence to the importance of antimicrobial peptides in these animals’ primary host defense. Pleurocidin may thus prove beneficial in both aquaculture and human medicine. Acknowledgments—We thank Drs. Charles Bevins and Nancy Connell for a critical reading of the manuscript, and Jim Jetko for histotechnological expertise. REFERENCES 1. Boman, H. G. (1995) Annu. Rev. Immunol. 13, 61–92 2. Murphy, C. J., Foster, B. A., Mannis, M. J., Selsted, M. E., and Reid, T. W. (1993) J. Cell. Physiol. 155, 408 – 413 3. Territo, M. C., Ganz, T., Selsted, M. E., and Lehrer, R. (1989) J. Clin. Invest. 84, 2017–2020 4. Boman, H. G. (1991) Cell 65, 205–207 5. Lehrer, R. I., Ganz, T., and Selsted, M. E. (1991) Cell 64, 229 –230 6. Zasloff, M. (1992) Curr. Opin. Immunol. 4, 3–7 7. Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W. L., and Bevins, C. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3952–3956 8. Jones, D. E., and Bevins, C. L. (1992) J. Biol. Chem. 267, 23216 –23225 9. Moore, K. S., Bevins, C. L., Brasseur, M. M., Tomassini, N., Turner, K., Eck, H., and Zasloff, M. (1991) J. Biol. Chem. 266, 19851–19857

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