Plicatamide, an Antimicrobial Octapeptide from Styela plicata Hemocytes

Abstract

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc⌬DOPA), where dc⌬DOPA represents decarboxy-(E)-␣,␤-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 dc⌬DOPA 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-HisTyr-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-dc⌬DOPA (plicatamide) 1 is a modified octapeptide found in the hemocytes of Styela plicata (1). In the preceding sequence, dc⌬DOPA indicates decarboxy-(E)-␣,␤-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. MATERIALS AND METHODS Peptide PurificationNative plicatamide was purified from freshly harvested hemocytes (blood cells) of S. plicata as described recently (1). 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. Peptide SynthesisThe synthetic peptides used in our initial experiments were customsynthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at Research Genetics (Huntsville, AL) and puri...

Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans

“…Therefore, peptides Sm1, Sm2, Sm3, Sm5, Sm6, and Sm7, as well as peptide Sm4, were regarded in the present study as the pool of S. mutans targeting vectors for library 1 STAMP construction. For the antimicrobial component, we selected PL-135, a short peptide based on an AMP isolated from tunicates (24) Activity of STAMP library 1. To roughly gauge STAMP antimicrobial activity and S. mutans selectivity, MIC assays were conducted with S. mutans and a panel of bacteria, including two oral Streptococcus species, S. sanguinis and S. sobrinus (Table 2).…”

Antimicrobial Peptides from Marine Invertebrates

“…Interestingly, a tachyplesin peptide derivative with a C-terminal extension of glycine-lysine was found in the hemocytes of Carcinoscorpius rotundicauda, which appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form (66). Naturally occurring peptides containing amidated C termini have been reported in antimicrobial peptides, polypeptide toxins, and sarcotoxins previously and may impart proteolytic resistance, as well as contribute to an increased overall positive charge (39,49,87). Like tachyplesin, the porcine neutrophil peptides, protegrins, are composed of 16 to 18 amino acid residues and contain two intramolecular cysteine disulfide bonds (38).…”

“…Styelin D displayed activity against gram-negative and -positive bacteria in 200 mM NaCl, the role of the extensive posttranslational modifications possibly being to preserve activity under low-pH or high-salt conditions in which an unmodified synthetic analogue was considerably less active (84). Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc⌬DOPA), in which dc⌬DOPA is decarboxy-(E)-␣,␤-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the ascidian Styela plicata (86,87). Wild-type and methicillin-resistant Staphylococcus aureus respond to plicatamide exposure with a massive potassium efflux that begins within seconds.…”

“…Typically, one expects such peptides to be cationic and amphipathic molecules with 16 to 40 residues (87), although two bovine peptides, dodecapeptide and indolicidin, are 12 to 13 amino acids long. In addition, plicatamide and other ascidian peptides (4,(85)(86)(87), including the halocyamines (below), are characterized by an oxidatively decarboxylated aromatic C-terminal residue. This structural feature may represent an alternative to C-terminal amidation for conferring proteolytic resistance and removal of the carboxylate's negative charge.…”

Elevated Expression of Paneth Cell CRS4C in Ileitis-prone SAMP1/YitFc Mice

“…The release or efflux of potassium ions from bacterial cells indicates that cell membrane physiology is disrupted and that the death of the organism has occurred (47,48,60). Accordingly, we also assayed efflux of bacterial cell K ϩ as an index of membrane-permeabilizing mechanisms of the CRS4C and Crp-4 peptides.…”