(Received for publication, December 20, 1996, and in revised form, February 25, 1997)
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 -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).
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
-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 pore-forming 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 -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.
Winter flounder epidermis and mucus extracts, isolated by 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 × 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 (7). 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 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-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.
Protein CharacterizationHeat 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 (29).
Peptide Synthesis and Antibody ProductionPleurocidin 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 ConditionsE. 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 AnalysisThe 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 (30), the agar-based assays used in this study quantified remaining viable colonies and not antimicrobial activity.
Kinetic Analysis1 × 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 ×, 1 ×, and 2 × 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 using LB without NaCl (10 g of tryptone, 5 g of yeast extract) and adding NaCl to cultures at increasing concentrations. 1 × 103 E. coli colony-forming units were initially incubated for 4 h at 37 °C with varying concentrations of pleurocidin (0-5.6 µM) and NaCl (0-1.25 M). Reactions were plated on LB agar plates and incubated overnight at 37 °C.
ImmunohistochemistryFresh skin sections were obtained, immediately fixed in 4% buffered paraformaldehyde for 4 h, dehydrated, and embedded in paraffin. Serial cross sections (5 µm) 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.
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 µg/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 -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 -helical conformation, indicating hydrophobic and
hydrophilic residues on opposing sides of a 20-amino 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 Gram-negative 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).
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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 × MIC (7.0 µM), antibacterial action increased dramatically after 15 min, and at 30 min only a few colony-forming units remained. At 1 × MIC (3.5 µM), antibacterial action was noted after 30 min with few colony-forming units at 120 min. At 0.5 × MIC (1.7 µM), 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-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 mucin-containing 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.
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 antimicrobial peptides (14, 17,
33). It is predicted to form an amphipathic -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 innate
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 -helical conformation, with hydrophobic and hydrophilic
sides segregating themselves.
Both dermaseptins (14, 17, 38) and ceratotoxins (31, 33) are
hypothesized to form an amphipathic -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
-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 patients 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 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 Gram-positive 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.
This report is New Jersey Sea Grant Publication No. NJSG-96-394.
We thank Drs. Charles Bevins and Nancy Connell for a critical reading of the manuscript, and Jim Jetko for histotechnological expertise.