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 againstEscherichia 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 α-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 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 againstEscherichia 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 α-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 (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar). 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 (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar). 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 (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar). In addition to microbicidal capabilities, certain peptides also confer diverse functions such as promotion of wound healing (2Murphy C.J. Foster B.A. Mannis M.J. Selsted M.E. Reid T.W. J. Cell. Physiol. 1993; 155: 408-413Google Scholar) and stimulation of monocyte chemotaxis (3Territo M.C. Ganz T. Selsted M.E. Lehrer R. J. Clin. Invest. 1989; 84: 2017-2020Google Scholar). While research has shown that vast quantities of antimicrobial peptides are found in inflammatory cells of most species studied (reviewed in Refs. 4Boman H.G. Cell. 1991; 65: 205-207Google Scholar, 5Lehrer R.I. Ganz T. Selsted M.E. Cell. 1991; 64: 229-230Google Scholar, 6Zasloff M. Curr. Opin. Immunol. 1992; 4: 3-7Google Scholar), recent interest has been directed toward the mucosal epithelia (7Diamond G. Zasloff M. Eck H. Brasseur M. Maloy W.L. Bevins C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3952-3956Google Scholar, 8Jones D.E. Bevins C.L. J. Biol. Chem. 1992; 267: 23216-23225Google Scholar, 9Moore K.S. Bevins C.L. Brasseur M.M. Tomassini N. Turner K. Eck H. Zasloff M. J. Biol. Chem. 1991; 266: 19851-19857Google Scholar, 10Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Google Scholar) (as reviewed in Ref. 11Bevins C.L. Ciba Found. Symp. 1994; 186: 250-260Google Scholar). 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 (12Samakovlis C. Kylsten P. Kimbrell D.A. Engstrom A. Hultmark D. EMBO J. 1991; 10: 163-169Google Scholar); magainin, from granular glands of Xenopus laevis (9Moore K.S. Bevins C.L. Brasseur M.M. Tomassini N. Turner K. Eck H. Zasloff M. J. Biol. Chem. 1991; 266: 19851-19857Google Scholar, 13Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Google Scholar); dermaseptin, from the skin of the arboreal frog Phyllomedusa bicolor (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar); tracheal antimicrobial peptide, from the columnar epithelial cells of the bovine trachea (7Diamond G. Zasloff M. Eck H. Brasseur M. Maloy W.L. Bevins C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3952-3956Google Scholar); and enteric defensins in the mammalian gastrointestinal tract (10Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Google Scholar, 15Lee J.Y. Boman A. Sun C.X. Andersson M. Jornvall H. Mutt V. Boman H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9159-9162Google Scholar). Frog skin produces a number of biologically active peptides including magainin (16Bevins C.L. Zasloff M. Annu. Rev. Biochem. 1990; 59: 395-414Google Scholar) and dermaseptin (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, 17Mor A. Amiche M. Nicolas P. Biochemistry. 1994; 33: 6642-6650Google Scholar). These linear peptides were shown to be antibacterial, antifungal, and antiprotozoal (17Mor A. Amiche M. Nicolas P. Biochemistry. 1994; 33: 6642-6650Google Scholar, 18Zasloff M. Martin B. Chen H.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 910-913Google Scholar). Both types of antimicrobial peptides have been shown to adopt an amphipathic α-helical conformation in hydrophobic media (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, 19Matsuzaki K. Harada M. Funakoshi S. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1991; 1063: 162-170Google Scholar, 20Jackson M. Mantsch H.H. Spencer J.H. Biochemistry. 1992; 31: 7289-7293Google Scholar). It has been suggested that this structural type of peptide binds anionic phospholipid-rich membranes similar to bacterial membranes and dissolves them like detergents (21Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Google Scholar, 22Gazit E. Boman A. Boman H.G. Shai Y. Biochemistry. 1995; 34: 11479-11488Google Scholar, 23Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Google Scholar). Natural antibiotics, which are not structurally homologous to magainin, dermaseptin, or ceratotoxin, have been isolated from several aquatic species. Pardaxin, a 33-amino acid pore-forming polypeptide toxin originally deemed a shark repellent peptide from sole (reviewed in Shai (24Shai Y. Toxicology. 1994; 87: 109-129Google Scholar)), has recently been determined to exert antibacterial activity (25Oren Z. Shai Y. Eur. J. Biochem. 1996; 237: 304-310Google Scholar). This peptide possesses a helix-hinge-helix structure and, unlike amphipathic α-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 (26Lemaitre C. Orange N. Saglio P. Saint N. Gagnon J. Molle G. Eur. J. Biochem. 1996; 240: 143-149Google Scholar). 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 (27Moore K.S. Wehrli S. Roder H. Rogers M. Forrest Jr., J.N. McCrimmon D. Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1354-1358Google Scholar, 28Wehrli S.L. Moore K.S. Roder H. Durell S. Zasloff M. Steroids. 1993; 58: 370-378Google Scholar). 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. Winter flounder epidermis and mucus extracts, isolated by scraping, were homogenized in a solution of 50 ml of 0.2 msodium acetate, 0.2% Triton X-100, 1 mmphenylmethylsulfonyl fluoride. The homogenate was centrifuged for 20 min at 20,000 × g (Beckman JA-17 rotor), applied to SepPak Vac 1g C18 cartridges (surface pH 7.0, 12% carbon, 12.5-nm pore size, 80-μm 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 (7Diamond G. Zasloff M. Eck H. Brasseur M. Maloy W.L. Bevins C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3952-3956Google Scholar). Fractions displaying a microbicidal zone of clearing were pooled and subjected to strong Poly-LC polysulfoethyl (5-μm particle size, 4.6 × 200 mm) cation-exchange HPLC 1The abbreviations used are: HPLC, high performance liquid chromatography; MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration. (linear AB gradients where A is 25% acetonitrile, 5 mm KH2PO4, pH 5.0, and B is 25% acetonitrile, 5 mmKH2PO4, 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-μm 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. 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-of-flight 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 (29Genetics Computer Group (1995) 8th Ed., Genetics Computer Group, Inc., Madison, WI.Google Scholar). 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. 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 aquatilisfrom 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. 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 × 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 μl 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 (30Kondejewski L.H. Farmer S.W. Wishart D.S. J. Biol. Chem. 1996; Scholar), the agar-based assays used in this and not antimicrobial activity. 1 × 103 E. coli colony-forming units were at 37 for min to with one of of pleurocidin 1 and × The were by on LB and were at 37 °C. determine concentration for pleurocidin activity, a MIC was performed with E. LB g of 5 g of and to at 1 × 103 E. coli colony-forming units were for at 37 with of pleurocidin and were on LB agar and at 37 °C. skin were in for and in of flounder skin were in and Briefly, were with and at of primary rabbit polyclonal or affinity-purified polyclonal in at °C. with were with by activity was with solution as for The was min of were for 5 min in was carried out with a using secretions of winter flounder were assayed for antimicrobial peptides on methods for similar in M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Google Scholar). from by scraping, were in This was and subjected to 1 A a absorbance Sephadex G-50 chromatography and antimicrobial activity in These to the peptide when by not these were pooled and subjected to ion HPLC, a single peak which eluted at 25.7 min 1 was determined to contain the antimicrobial activity. This was purified by reversed-phase HPLC, which in a single peak at 29.1 min 1 which exhibited antimicrobial activity. on both the ion and reversed-phase HPLC this antimicrobial peptide which are similar to of magainin and Antimicrobial activity was when the fraction was subjected to boiling for 5 min. with 20 proteinase K for 30 min at 37 that antimicrobial activity is not The microbicidal reversed-phase HPLC resuspended in water, was subjected to mass analysis, which determined a single ion at of of sequence analysis, two amino acids to be amino acid HPLC analysis and mass spectrometry it was found that the two were a and on a mass by and amino acid analysis, the of these two amino acids Microchemistry The peptide sequence is in this peptide from the and is determined to be we named the peptide against indicated significant sequence to pleurocidin with the dermaseptin and ceratotoxin of antimicrobial peptides, which are shown in The and have been to amphipathic (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, D. R. R. Biochem. Biol. 1993; Scholar). was to hydrophobic and the structure of pleurocidin M. Biophys. J. Scholar). pleurocidin in an amphipathic α-helical hydrophobic and on of a acid of the pleurocidin that the is cationic in Pleurocidin was tested against 11 Gram-positive and Gram-negative bacteria for bactericidal and bacteriostatic activity. were which both mucosal and bacteria were A. salmonicida from skin of C. from gills of diseased and mucor eggs of winter that this peptide is active against both Gram-negative and Gram-positive bacteria. Most we that E. coli and B. are most to pleurocidin S. marcescens, and P. are most In the bacteriostatic concentration (MIC) of pleurocidin is to the bactericidal concentration (MBC) of this peptide for A. which possesses a bactericidal and bactericidal activity of pleurocidin against 11 different and salmonicida typhimurium typhimurium II in a determine the of bactericidal activity of pleurocidin, a was performed using E. coli We a mechanism of action with to microbicidal activity. × MIC antibacterial action min, and at 30 min a colony-forming units 1 × MIC antibacterial action was 30 min with colony-forming units at min. × MIC antimicrobial action min of and most of the bacteria were not with pleurocidin for min. that the of colony-forming with pleurocidin concentration. Winter flounder from of approximately in winter to in determine the microbicidal action of pleurocidin is on concentration was with of pleurocidin and to in bacteriostatic and bactericidal as described for We determined that pleurocidin is at physiological pleurocidin bacteria to mm NaCl, the concentration of in methods were performed to determine the of pleurocidin a of flounder skin. We determined that pleurocidin is in the epithelial mucous cells by using polyclonal antisera and affinity-purified with as a The in these cells is of pleurocidin in This is in to the of in the mucous cells of the of other epithelial cells and is in both the and as well as in the affinity-purified were to in was We report the discovery of pleurocidin, a 25-residue peptide with broad-spectrum antimicrobial activity, in the skin secretions of the winter flounder. Pleurocidin sequence homology with the dermaseptin and ceratotoxin of antimicrobial peptides (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, 17Mor A. Amiche M. Nicolas P. Biochemistry. 1994; 33: 6642-6650Google D. M. R. J. Biol. Chem. 1995; Scholar). It is predicted to an amphipathic α-helical structure similar to many other antimicrobial peptides, which exert their function by in the bacterial (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, 19Matsuzaki K. Harada M. Funakoshi S. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1991; 1063: 162-170Google Scholar, 20Jackson M. Mantsch H.H. Spencer J.H. Biochemistry. 1992; 31: 7289-7293Google D. Zasloff M. A. 1988; Scholar, H.C. J.H. 1988; Scholar, R. K. T. Zasloff M. D. Biochemistry. 1990; Scholar). Pleurocidin microbicidal action against a wide range of both Gram-negative and Gram-positive bacteria from aquatic species broad-spectrum antibacterial This that like several other aquatic possess innate host defense to combat microbes on their mucosal a bacterial species to the of winter flounder is to This a bacterial mechanism that has to the of antimicrobial peptides. Antimicrobial peptides are to exert action by to the of membranes and a lysis of the for in solution with a structure Zasloff M. Ciba Found. Symp. 1994; 186: Scholar). It is the of magainin to an that the peptide to assume an amphipathic α-helical with hydrophobic and Both (14Mor A. Nguyen V.H. Delfour A. Migliore-Samour D. Nicolas P. Biochemistry. 1991; 30: 8824-8830Google Scholar, 17Mor A. Amiche M. Nicolas P. Biochemistry. 1994; 33: 6642-6650Google Scholar, A. K. Nicolas P. J. Biol. Chem. 1994; Scholar) and D. R. R. Biochem. Biol. 1993; Scholar, D. M. R. J. Biol. Chem. 1995; Scholar) are to an amphipathic in the that a bacterial approximately amino acids in length. the primary of pleurocidin, dermaseptin, and ceratotoxin are it is that their structure also be when these peptides are found to with the bacterial α-helical peptides, such as dermaseptin, ceratotoxin, and magainin, have been suggested to anionic phospholipid-rich membranes similar to bacterial and them like detergents (21Pouny Y. Rapaport D. Mor A. Nicolas P. Shai Y. Biochemistry. 1992; 31: 12416-12423Google Scholar, 22Gazit E. Boman A. Boman H.G. Shai Y. Biochemistry. 1995; 34: 11479-11488Google Scholar, 23Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Google Scholar). This of action may be of a between concentration of peptide, and bactericidal activity. This has been with other antimicrobial peptides. of this the concentration of to determine winter flounder on the their from to It was determined that concentration not have an on antibacterial inhibited the microbicidal of pleurocidin at the high that high bactericidal activity was a of Cell. 1996; 85: Scholar) recently discovered that the high concentration in the of to a to bacteria, and in the of pleurocidin bactericidal action at high well the of this antimicrobial peptide may in more antibiotic for The structure and function of pleurocidin from other of antimicrobial molecules in marine species. Pleurocidin is in the epithelial mucous cells of the is found in mucous glands that line the and of sole E. Natl. Scholar). antibacterial proteins are kDa) than pleurocidin and the is (26Lemaitre C. Orange N. Saglio P. Saint N. Gagnon J. Molle G. Eur. J. Biochem. 1996; 240: 143-149Google Scholar). Both proteins ion channels in the bacterial in a similar to it has been shown that antibacterial such as A from the N. T. K. H. 1995; Scholar) and from the R. H. N. Y. T. T. Eur. J. Biochem. 1992; Scholar), are than stimulation from a concentration of pleurocidin the mucous that this antimicrobial peptide is a stimulation from the cells. This is with magainin to the granular glands in the skin and Biochem. J. 1987; Scholar). analysis that pleurocidin is in the mucous cells of the flounder skin. may be in is through the in A and a of of mucous cell It has to be determined this is mass of the antimicrobial peptide or an of tissue and certain a at the epithelial of skin. This may be to found on the of the skin which have been suggested to defense and in mucous including pleurocidin, to the skin Cell Scholar). In we have isolated and a novel 25-residue antimicrobial peptide, pleurocidin, from the skin of winter flounder with the amino acid sequence This peptide has been shown to exert activity against a wide range of Gram-positive and Gram-negative bacteria. Pleurocidin has been to the epithelial mucous cells of the flounder skin. It has high amino acid sequence homology with the dermaseptin and ceratotoxin of antimicrobial peptides. This which is diverse to the of antimicrobial peptides in these primary host defense. Pleurocidin may in both and We Bevins and for a of the and for