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Plicatamide, an Antimicrobial Octapeptide from Styela plicata Hemocytes

2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês

10.1074/jbc.m211332200

1083-351X

J. Andy Tincu, Lorenzo P. Menzel, Rustam Azimov, Jennifer Sands, Teresa Hong, Alan J. Waring, Steven W. Taylor, Robert I. Lehrer,

Aquaculture disease management and microbiota

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dcΔDOPA), where dcΔDOPA represents decarboxy-(E)-α,β-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dcΔDOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design. Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dcΔDOPA), where dcΔDOPA represents decarboxy-(E)-α,β-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dcΔDOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design. Phe-Phe-His-Leu-His-Phe-His-dcΔDOPA decarboxy-(E)-α,β-dehydro-3,4-dihydroxyphenylalanine 3,4-dihydroxyphenylalanine methicillin-resistant S. aureus colony-forming units phosphate-buffered saline Fourier transform infrared minimal effective concentration 4-morpholineethanesulfonic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide high pressure liquid chromatography area under the curve N-β-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine polymorphonucleated granulocytes Phe-Phe-His-Leu-His-Phe-His-dcΔDOPA (plicatamide)1 is a modified octapeptide found in the hemocytes of Styela plicata (1Tincu J.A. Craig A.G. Taylor S.W. Biochem. Biophys. Res. Commun. 2000; 270: 421-424Crossref PubMed Scopus (23) Google Scholar). In the preceding sequence, dcΔDOPA indicates decarboxy-(E)-α,β-dehydro-3,4-dihydroxyphenylalanine. Although the sequence of plicatamide did not resemble a conventional antimicrobial peptide, we examined its antimicrobial properties because hemocytes are key participants in innate antimicrobial defenses. Despite its small size, plicatamide proved to be a potent, rapidly acting, and broad spectrum antimicrobial. We also prepared the following four synthetic analogues that differed from plicatamide only in their C-terminal residue: tyrosine amide in PL-101; tyrosine acid in PL-102; DOPA (3,4-dihydroxyphenylalanine) acid in PL-103; and DOPA-amide in PL-104. Of these octapeptides, PL-101 most closely simulated the antimicrobial properties of native plicatamide. This report will describe the effects of plicatamide on staphylococci. Native plicatamide was purified from freshly harvested hemocytes (blood cells) of S. plicata as described recently (1Tincu J.A. Craig A.G. Taylor S.W. Biochem. Biophys. Res. Commun. 2000; 270: 421-424Crossref PubMed Scopus (23) Google Scholar). We determined their peptide content either by performing quantitative amino acid analysis or by doing analytical reverse phase-HPLC on a C18 column and then computing and comparing the area under the curve (AUC) at 215 nm with the AUC of an appropriate standard previously subjected to quantitative amino acid analysis. The synthetic peptides used in our initial experiments were custom-synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at Research Genetics (Huntsville, AL) and purified to homogeneity by reverse phase-HPLC. Mushroom tyrosinase (6680 units/mg) was purchased from Sigma, and all other reagents were of analytical grade. Mushroom tyrosinase (Sigma) was used to prepare PL-103 and -104 (Table I) by converting the C-terminal tyrosine of PL-101 and -102 to DOPA (2Craig A.G. Taylor S.W. J. Am. Soc. Mass Spectrom. 2001; 12: 470-474Crossref PubMed Scopus (6) Google Scholar, 3Taylor S.W. Anal. Biochem. 2002; 302: 70-74Crossref PubMed Scopus (40) Google Scholar). Briefly, the synthetic peptides (1 mg/ml, final concentration) were dissolved in 10 ml of 20 mm borate, 0.1m phosphate/ascorbate buffer, pH 7.0, in a plastic reaction vessel. Before starting the reaction by adding 100 μg/ml (final concentration) of mushroom tyrosinase (Sigma, 6680 units/mg), we removed a 50-μl aliquot and acidified it with 2 μl of 6n HCl. Tyrosinase reactions were run at room temperature under a stream of humidified air. Aliquots of the reaction mixture were removed every 20 min and subjected to analytical reverse phase-HPLC to monitor the progress of the reaction. This chromatography was performed over 50 min on a Phenomenex Jupiter Series 4.6 × 250-mm analytical C-18 column (10 μm, 300-Å pore size), using a 0–40% linear gradient of water with 0.1% trifluoroacetic acid to acetonitrile in 0.085% trifluoroacetic acid. After 60 min, the reaction was terminated by adding 200 μl of 6 n HCl, and the mixture was desalted by loading it directly onto a Sep-Pac Vac 1-g (6 ml) cartridge (Waters Associates, Milford, MA). After washing the cartridge with 20 ml of water containing 0.1% trifluoroacetic acid, the peptides were eluted with 10 ml of 60% acetonitrile containing 0.085% trifluoroacetic acid. Subsequent purification was obtained by multiple runs on a 10 × 250-mm C-18 reverse phase-HPLC column. The peptide sequences were checked by tandem mass spectrometry on a Finnigan LCQ Ion Trap Instrument. Subsequent batches of PL-101 were synthesized in our UCLA laboratory on an ABI 433A peptide synthesizer using FastMocTM chemistry and purified by reverse phase-HPLC as described above.Table IPeptides used in this studyPeptideResidues 1–7Residue 8PlicatamideFFHLHFHdcΔDOPAPL-101FFHLHFHTyrosine amidePL-102FFHLHFHTyrosine acidPL-103FFHLHFHDOPA acidPL-104FFHLHFHDOPA amidePlicatamide was purified from the hemocytes of S. plicata.All other peptides were synthetic and were prepared as described in the text. Open table in a new tab Plicatamide was purified from the hemocytes of S. plicata.All other peptides were synthetic and were prepared as described in the text. The assay has been described elsewhere (4Lehrer R.I. Rosenman M. Harwig S.S. Jackson R. Eisenhauer P. J. Immunol. Methods. 1991; 137: 167-173Crossref PubMed Scopus (583) Google Scholar). Our Gram-positive test organisms wereStaphylococcus aureus 930918-3, MRSA ATCC 33591, a methicillin-resistant S. aureus strain, and Listeria monocytogenes, strain EGD. In some experiments we also testedEscherichia coli, ML-35p, and Pseudomonas aeruginosa, MR3007, a strain that was resistant to many conventional antibiotics. Native plicatamide was serially 3.16-fold diluted with 0.01% acetic acid that contained 0.1% human serum albumin to minimize its nonspecific adsorption to plastic tubes. Organisms were grown to mid-logarithmic phase at 37 °C in trypticase soy broth. After they were washed with 10 mmphosphate buffer, pH 7.4, ∼4 × 106 bacterial colony-forming units (CFU) were incorporated into 10 ml of the underlay gel mixture. Unless otherwise stated, the underlay gels also contained 1% w/v agarose (Sigma A-6013), 10 mm sodium phosphate buffer, pH 7.4, and 0.3 mg/ml trypticase soy broth powder. Some underlay gels were supplemented with 100, 175, or 250 mmNaCl. A 6 × 6 array of sample wells, each 3.2 mm in diameter and 1.2 mm deep, was punched in the underlay gel. These allowed 8-μl aliquots of each dilution to be introduced. After the plates had incubated for 3 h at 37 °C, a nutrient-rich overlay gel (60 mg/ml trypticase soy broth powder in 1% v/v agarose) was poured, and the incubation was continued overnight to allow surviving organisms to form microcolonies. The diameters of completely clear zones were measured to the nearest 0.1 mm and expressed in units (1 unit = 0.1 mm), after first subtracting the well diameter. Because a linear relationship exists between the zone diameter and the log10of the peptide concentration, the X intercept of this line was determined by a least mean squares fit and was considered to represent the minimal effective concentration (MEC). Stationary or mid-logarithmic phase bacteria were prepared as described above and incubated with antimicrobial peptides at 37 °C in an agarose-free liquid medium containing 100 mm NaCl, 10 mm sodium phosphate, or Tris buffer, pH 7.4, and such other additions as are described in the text. Aliquots (20 μl) were removed at intervals, diluted appropriately, and transferred to nutrient agar plates with a Spiral Plater (Spiral Biotech, Rockville, MD). Colonies were counted after overnight incubation at 37 °C. These assays used cation-adjusted, Mueller Hinton II Broth (BD Biosciences) and were performed according to the guidelines of the National Committee for Clinical Laboratory Standards (5National Committee for Clinical Laboratory Standards Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. National Committee for Clinical Laboratory Standards, Wayne, PA1993Google Scholar), except that the 10× stock plicatamide was prepared and serially diluted in acidified water (sterile 0.01% acetic acid) instead of in Mueller Hinton II Broth. Test organisms were incubated overnight in 50 ml of trypticase soy broth at 37 °C, washed three times with buffer (100 mm NaCl, 10 mm Tris acetate, pH 7.4), and resuspended in this buffer at ∼2.5 × 108 CFU/ml, based on A620. Experiments were done at 37 °C in stirred polypropylene tubes surrounded by a 50-ml water-jacketed reaction vessel (Kimble/Kontes, Vineland, NJ). The tube contained 6 × 107 CFU of washed, stationary phase bacteria in 100 mm NaCl, 10 mm Tris acetate, pH 7.4, in a final volume of 250 μl. An Orion SensorLink PCM-700 pH/ISE meter, fitted with a MI-442 potassium electrode (Microelectrodes, Bedford, NH) and an SDR-2 reference electrode (World Precision Instruments, Sarasota, FL), was used as described previously (6Orlov D.S. Nguyen T. Lehrer R.I. J. Microbiol. Methods. 2002; 49: 325-328Crossref PubMed Scopus (80) Google Scholar). Oxygen consumption by washed, stationary phase bacteria was measured with a Clark-type oxygen electrode (Hansatech Ltd., Norfolk, UK). Briefly, an overnight culture of MRSA was twice washed with PBS and adjusted to 3 × 108 CFU/ml in this medium. After adding bacteria (1 ml) to the continuously stirred chamber, we added 10 μl of full strength trypticase soy broth, and we measured the basal rate of O2 consumption at room temperature for 5–10 min before adding peptide (10 μg/ml final concentration). The cytotoxicity of plicatamide for ME-180 (ATCC HTB-33) human cervical epithelial target cells was assessed with an MTT-tetrazolium reduction assay (Roche Molecular Biochemicals). Target cells were grown to confluency in RPMI 1640 medium with 10% fetal bovine serum and 50 μg/ml gentamicin and harvested with trypsin/EDTA. After washing them with this medium, their concentration and viability (trypan blue exclusion) was determined, and they were suspended at 5 × 104 cells/ml. Cell aliquots (100 μl) were dispensed into 96-well tissue plates (Corning Glass) and incubated for 5 h at 37 °C in room air with 5% CO2 before the peptides were added. After 20 additional hours of incubation, we added 10 μl of MTT solution, followed 4 h later by 100 μl of extraction buffer. After overnight extraction of the reduced MTT-tetrazolium, absorbance was measured at 600 and 650 nm, on a Spectramax 250 spectrophotometer (Molecular Devices, Sunnyvale, CA). Hemolytic activity was tested by incubating various concentrations of peptide with a suspension (2.8% v/v) of washed human or sheep red cells in Dulbecco's phosphate-buffered saline. After 30 min at 37 °C, the tubes were centrifuged, and the absorbance (A) of the supernatants was measured. The percentage of hemolysis was calculated by Equation 1, whereAexper and Acontrolsignify the absorbance values of supernatants from treated and untreated red cells, and Atotal is the supernatant of red cells treated with 0.1% Triton X-100.%release=((Aexper−Acontrol)/(Atotal−Acontrol))×100Equation 1 For transmission electron microscopy, 5 × 108bacterial CFU/ml were exposed at room temperature to 42.5 μg/ml native plicatamide in PBS (100 mm NaCl and 10 mm sodium phosphate, pH 7.4) containing 1% v/v trypticase soy broth. At intervals, 1-ml aliquots were removed, centrifuged briefly at 2000 × g, and immediately resuspended in 1 ml of freshly made 2% glutaraldehyde in PBS. After 30 min on ice, the fixed organisms were washed in PBS. For scanning EM, 10% of the above bacteria were adhered for 30 min to mixed cellulose ester membrane filters with 0.025-μm pores (Millipore, Bedford, MA). The filters were washed twice with 10 mm sodium phosphate, pH 7.4, and dehydrated through a graded ethanol series into hexamethyldisilane. After carbon coating, the samples were viewed on a Cambridge Stereoscan Electron Microscope. The remaining bacteria were washed in PBS, post-fixed for 45 min at room temperature in 1% osmium tetroxide, dehydrated through ethanol to propylene oxide, and embedded in Epon 812. After staining with uranyl acetate at 60 °C for 15 min, and then by lead citrate, the sections were viewed on a JEOL CX II microscope. Solvent-containing phospholipid bilayer membranes were formed by placing a small bubble of 15 mg/ml lipid solution inn-heptane onto the end of Teflon tubing with 0.25-mm inner diameter. The design of the chamber allowed 50 μl of solution to be rapidly introduced immediately adjacent to the membrane (7Mirzabekov T.A. Silberstein A.Y. Kagan B.L. Methods Enzymol. 1999; 294: 661-674Crossref PubMed Scopus (26) Google Scholar). E. coli total lipid extract were purchased from Avanti Polar Lipids (Alabaster, AL) and stored at −20 °C. Agar salt bridges connected the electrodes to the solutions, and voltage clamp conditions were employed in all experiments. Thecis-side (i.e. the side to which peptide was added) was taken as ground. All stated voltages refer to the voltage of the trans-side. Current was recorded with an Axopatch-1C amplifier with a CV-3B head stage and stored on videotape for later playback and analysis. Membrane capacitance and resistance were monitored to ensure the formation of reproducible membranes. The peptide stock solution (2 mg/ml) was stored at 4 °C, and the working solutions were prepared immediately before use. The bath solution contained 100 mm KCl and 10 mm Tris-HCl buffer, pH 7.4, or 10 mm MES-Tris buffer, pH 5.5 and pH 6.5, or 10 mm Tris citrate buffer pH 7.4. Fig.1a summarizes a series of radial diffusion assays done in underlay gels containing 100 mm NaCl at pH 7.4 and pH 5.5. Both native plicatamide and PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide) were more effective microbicides at neutral pH. Although native plicatamide and PL-101 had similar potency against the two Gram-positive organisms, S. aureus and L. monocytogenes, the native plicatamide was 2–3-fold more potent against the Gram-negative test strains, E. coliand P. aeruginosa. Fig. 1b shows that PL-101 was considerably more active than either PL-102 (Phe-Phe-His-Leu-His-Phe-His-Tyr-acid) or the two DOPA-containing peptides PL-103 and PL-104. The sequences of these peptides are shown in Table I. In several of our preparations of native plicatamide, FTIR analyses revealed additional bands, characteristic of lipids and/or phospholipid, in addition to the expected absorption bands for a peptide (Fig.2a). A mixture of synthetic PL-101 and palmitoyloleoylphosphatidylglycerol provided a similar FTIR spectrum (Fig. 2b), whereas PL-101 gave a typical peptide spectrum (Fig. 2c). Because the antimicrobial data shown in Fig. 1a were obtained with a preparation of plicatamide that contained only the expected peptide bands, we consider it unlikely that any co-purified lipids were responsible for the antimicrobial properties of our other preparations. Fig. 3 shows the results of experiments comparing the effects of pH and salinity on the antimicrobial activity of plicatamide and PL-101. These native plicatamide preparations did contain associated (phospho)lipids by FTIR. Again, native plicatamide and synthetic PL-101 were substantially more effective at pH 7.4, than at pH 5.5 despite their greater cationicity at the lower pH. The MEC of plicatamide in 100 mm NaCl at pH 7.4 ranged from 1.0 to 2.5 μg/ml for E. coli, S. aureus, and L. monocytogenes. These results are quite similar to those obtained with the phospholipid-free preparation of plicatamide (Fig.1a). We obtained similar MEC values when the underlay gels contained 250 μg/ml (data not shown). Colony counting experiments revealed that native plicatamide killed MRSA and S. aureus very rapidly (Fig.4). The peptide was equally effective in medium with or without nutrients, and we found little difference in the susceptibility of mid-logarithmic and stationary phase staphylococci to plicatamide (data not shown). Furthermore, staphylocidal activity was not impaired by including 10 μg/ml catalase in the medium, nor did inclusion of 1 mm Ca2+ or 1 mmMg2+ impair it (data not shown). We assessed the membrane integrity of plicatamide-treated staphylococci by measuring their loss of cytoplasmic potassium (Fig.5). To ensure adequate amounts (100–200 nmol) of total K+, bacterial concentrations of ∼7.5 × 107 CFU/ml were used. We also measured viability by removing aliquots at intervals for colony counting. The virtually immediate and substantial efflux of K+ from plicatamide-treated MRSA is consistent with an antimicrobial mechanism that targets their cell membrane. Native plicatamide induced a similar efflux of K+ from S. aureus, and synthetic PL-101 induced K+ efflux from both S. aureus and MRSA (data not shown). We also examined the effects of plicatamide on planar bilayer membranes prepared from E. coli lipids dissolved in n-heptane. The untreated membranes were stable between ±100 mV, and displayed low ( 2–3 logs after 30 and 120 min of incubation (data not shown). A few additional experiments revealed that the few organisms that survived exposure to plicatamide could repopulate the culture, thereby masking the antimicrobial properties of plicatamide, at least for microbroth dilution assays. This effect is illustrated in Fig.8. MRSA treated with plicatamide showed many alterations. After only 5 min, striking changes were observed by scanning electron microscopy, wherein multiple small dome-shaped bulges, often arranged in linear and clustered arrays (Fig. 9), deformed their surfaces. These abnormalities became more marked as the duration of exposure to plicatamide increased (Fig. 10). In many bacteria, large amounts of cytoplasm extruded beyond the confines of the cell wall. Transmission electron microscopy of plicatamide-treated bacteria revealed fixed prominent mesosomes, even in cells fixed as early as 5 min after exposure to plicatamide (Fig.11). Many plicatamide-treated MRSA contained electron dense material between their plasma membrane and cell wall, representing partially contained "eruptions" of cytoplasm akin to the more flamboyant manifestations evident in Fig.10.Figure 10Later effects on morphology. More advanced surface deformities were noted in MRSA exposed to native plicatamide (15 μg/ml) for 15–60 min. In addition, many cells showed transmural extrusions of their cytoplasm.View Large Image Figure ViewerDownload (PPT)Figure 11Transmission electron microscopy. Many plicatamide-treated MRSA contained mesosomes (white arrows), and such structures were evident as early as 5 min post-exposure to plicatamide. After longer exposures to plicatamide (15 μg/ml), some MRSA contained electron dense material between their plasma membrane and thick wall.View Large Image Figure ViewerDownload (PPT) Plicatamide-lysed human erythrocytes, acting with almost the same potency as melittin on a weight/volume basis (Fig. 12). However, in marked contrast to melittin, plicatamide was not hemolytic for sheep red blood cells, even when applied at 80 μg/ml. Moreover, although melittin induced hemolysis over a broad pH range, the hemolytic properties of plicatamide were markedly diminished as acidity increased. Furthermore, whereas melittin was exceptionally cytotoxic for human cervical ME-180 epithelial cells, plicatamide was relatively noncytotoxic for these cells under the same conditions (data not shown). Plicatamide (Fig. 13) is an interesting peptide for many reasons, not the least of which is that it violates conventional notions about antimicrobial peptides. Typically, one expects such peptides to be cationic and amphipathic molecules with 16–40 residues (11Dathe M. Wieprecht T. Biochim. Biophys. Acta. 1999; 1462: 71-87Crossref PubMed Scopus (625) Google Scholar, 12Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1145) Google Scholar, 13Gennaro R. Zanetti M. Biopolymers. 2000; 55: 31-49Crossref PubMed Scopus (282) Google Scholar, 14Hancock R.E. Lancet Infect. Dis. 2001; 1: 156-164Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 15Tossi A. Sandri L. Giangaspero A. Biopolymers. 2000; 55: 4-30Crossref PubMed Scopus (1031) Google Scholar). A few smaller antimicrobial peptides with 11–13 residues have been described. These include the bactenecin dodecapeptides of bovine or ovine neutrophils (16Bagella L. Scocchi M. Zanetti M. FEBS Lett. 1995; 376: 225-228Crossref PubMed Scopus (101) Google Scholar, 17Romeo D. Skerlavaj B. Bolognesi M. Gennaro R. J. Biol. Chem. 1988; 263: 9573-9575Abstract Full Text PDF PubMed Google Scholar), bovine indolicidin (18Selsted M.E. Novotny M.J. Morris W.L. Tang Y.Q. Smith W. Cullor J.S. J. Biol. Chem. 1992; 267: 4292-4295Abstract Full Text PDF PubMed Google Scholar, 19Schibli D.J. Hwang P.M. Vogel H.J. Biochemistry. 1999; 38: 16749-16755Crossref PubMed Scopus (142) Google Scholar), and tigerinins, antimicrobial peptides isolated from the skin secretions of a frog, Rana tigerina (20Sai K.P. Jagannadham M.V. Vairamani M. Raju N.P. Devi A.S. Nagaraj R. Sitaram N. J. Biol. Chem. 2001; 276: 2701-2707Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Plicatamide contains eight residues, and it is only modestly cationic at pH 7.4, and when it was rendered more cationic (at pH 5.5) its activity decreased. To our knowledge, only two smaller antimicrobial peptides have been found in animals: 5-S-GAD, and halocyamine A.N-β-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD) is a pentapeptide that was purified from the hemolymph of injured or infected "fleshflies" (Sarcophaga peregrina) (21Leem J.Y. Nishimura C. Kurata S. Shimada I. Kobayashi A. Natori S. J. Biol. Chem. 1996; 271: 13573-13577Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Because the antimicrobial activities of 5-S-GAD were completely inhibited by catalase, it was suggested that H2O2 participates in its antimicrobial mechanism, as well as its induction of apoptosis in HL60 cells (22Hijikata M. Matsumoto H.N. Kobayashi A. Nifuji A. Noda M. Natori S. FEBS Lett. 1999; 457: 405-408Crossref PubMed Scopus (9) Google Scholar). Because we found that catalase did not inhibit the bactericidal effects of plicatamide, its antimicrobial mechanism evidently differs from that of 5-S-GAD. Other properties of the 5-S-GAD molecule include an ability to inhibit tyrosine phosphorylation of certain kinases, PTK p60(v-src) and PTK p210(BCR-ABL) (23Leem J.Y. Park H.Y. Fukazawa H. Uehara Y. Natori S. Biol. & Pharm. Bull. 1998; 21: 784-785Crossref PubMed Scopus (11) Google Scholar, 24Zheng Z.B. Nagai S. Iwanami N. Suh D.Y. Kobayashi A. Hijikata M. Natori S. Sankawa U. Chem. Pharm. Bull. 1999; 47: 136-137Crossref PubMed Scopus (5) Google Scholar). Halocyamine A is a tetrapeptide (histidyl-3,4-dihydroxyphenylalanine-glycyl-bromodidehydrotryptamine) that, like plicatamide, is also found in the hemocytes of a tunicate, in this case Halocynthia roretzi. Halocyamine A was reported to inhibit the growth of yeast and of the marine bacteriaAchromobacter aquamarinus and Pseudomonas perfectomarinus (25Azumi K. Yoshimizu M. Suzuki S. Ezura Y. Yokosawa H. Experientia (Basel). 1990; 46: 1066-1068Crossref PubMed Scopus (41) Google Scholar). Neither its antimicrobial mechanism nor the effects of catalase on its activity have been described. It is remarkable that three of the smallest known antimicrobial peptides (5-S-GAD, halocyamine A, and plicatamide) should all contain a DOPA moiety. Although this could be a coincidence, it may also be an indication that this residue plays an important functional role. Although PL-103 and PL-104, the DOPA-containing synthetic analogues of plicatamide examined here, were unimpressive microbicides, we have yet to prepare an exact synthetic replica of plicatamide. Another possible function of DOPA and dcΔDOPA might be