Lycotoxins, Antimicrobial Peptides from Venom of the Wolf SpiderLycosa carolinensis

Abstract

Two peptide toxins with antimicrobial activity, lycotoxins I and II, were identified from venom of the wolf spiderLycosa carolinensis (Araneae: Lycosidae) by virtue of their abilities to reduce ion and voltage gradients across membranes. Both peptides were purified to homogeneity by reversed-phase liquid chromatography and determined to have the following primary structures by Edman microsequencing: IWLTALKFLGKHAAKHLAKQQLSKL-NH2 for lycotoxin I and KIKWFKTMKSIAKFIAKEQMKKHLGGE-OH for lycotoxin II. The predicted secondary structures of the lycotoxins display amphipathic α-helix character typical of antimicrobial pore-forming peptides. Antimicrobial assays showed that both lycotoxins potently inhibit the growth of bacteria (Escherichia coli) and yeast (Candida glabrata) at micromolar concentrations. To verify its hypothesized pore-forming activity, lycotoxin I was synthesized and shown to promote efflux of Ca2+ from synaptosomes, to cause hemolysis of erythrocytes, and to dissipate voltage gradients across muscle membrane. The lycotoxins may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion. Spider venoms may represent a potentially new source of novel antimicrobial agents with important medical implications. Two peptide toxins with antimicrobial activity, lycotoxins I and II, were identified from venom of the wolf spiderLycosa carolinensis (Araneae: Lycosidae) by virtue of their abilities to reduce ion and voltage gradients across membranes. Both peptides were purified to homogeneity by reversed-phase liquid chromatography and determined to have the following primary structures by Edman microsequencing: IWLTALKFLGKHAAKHLAKQQLSKL-NH2 for lycotoxin I and KIKWFKTMKSIAKFIAKEQMKKHLGGE-OH for lycotoxin II. The predicted secondary structures of the lycotoxins display amphipathic α-helix character typical of antimicrobial pore-forming peptides. Antimicrobial assays showed that both lycotoxins potently inhibit the growth of bacteria (Escherichia coli) and yeast (Candida glabrata) at micromolar concentrations. To verify its hypothesized pore-forming activity, lycotoxin I was synthesized and shown to promote efflux of Ca2+ from synaptosomes, to cause hemolysis of erythrocytes, and to dissipate voltage gradients across muscle membrane. The lycotoxins may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion. Spider venoms may represent a potentially new source of novel antimicrobial agents with important medical implications. Most spiders are voracious predators, employing paralytic venoms to immobilize or kill their prey. The majority of known spider venom toxins attack the nervous system, causing disruption of impulse conduction and/or synaptic transmission through actions on ion channels (1Kawai N. Nakajima T. Chang L.W. Dyer R.S. Handbook of Neurotoxicology. Marcel Dekker, Inc., New York1995: 667-682Google Scholar, 2Adams M.E. Swanson G. Trends Neurosci. 1994; 19: 1-37Google Scholar, 3Nicholson G.M. Willow M. Howden M.E. Narahashi T. Pfluegers Arch. Eur. J. Physiol. 1994; 428: 400-409Crossref PubMed Scopus (74) Google Scholar) and exocytosis proteins (4Davletov B.A. Shamotienko O.G. Lelianova V.G. Grishin E.V. Ushkaryov Y.A. J. Biol. Chem. 1996; 271: 23239-23245Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 5Krasnoperov V.G. Beavis R. Chepurny O.G. Little A.R. Plotnikov A.N. Petrenko A.G. Biochem. Biophys. Res. Commun. 1996; 227: 868-875Crossref PubMed Scopus (77) Google Scholar). Alternatively, many spider venoms cause cell membrane disruption and consequent tissue necrosis through enzymatic actions (6Futrell J.M. Am. J. Med. Sci. 1992; 304: 261-267Crossref PubMed Scopus (247) Google Scholar, 7Tambourgi D.V. Magnoli F.C. von Eickstedt V.R. Benedetti Z.C. Petricevich V.L. da Silva W.D. J. Immunol. 1995; 155: 4459-4466PubMed Google Scholar, 8Barbaro K.C. Sousa M.V. Morhy L. Eickstedt V.R. Mota I. J. Protein Chem. 1996; 15: 337-343Crossref PubMed Scopus (47) Google Scholar). The wolf spider Lycosa carolinensis (Araneae: Lycosidae) is an omnivorous hunting spider with a widespread distribution in the United States. The spider hides underground during the day, but leaves its burrow during the night in search of prey. The effects of wolf spider bites on humans are not considered to be severe (9Ribeiro L.A. Jorge M.T. Piesco R.V. Nishioka S.D.A. Toxicon. 1990; 28: 715-717Crossref PubMed Scopus (38) Google Scholar), and relatively little is known with regard to the chemical composition ofL. carolinensis venom. Previous studies involving related species indicate the presence of neuroactive constituents (10Cruz J.S. Cotta G. Diniz C.R. Beirao P.S. Braz. J. Med. Biol. Res. 1994; 27: 2653-2659PubMed Google Scholar). Here we characterize two peptide toxins from L. carolinensisvenom that are both neuroactive and antimicrobial. Named here as “lycotoxins,” they appear from their amphipathic character and physiological actions to function as pore formers to increase membrane permeability and to effect lysis of both prokaryotic and eukaryotic cells. Wolf spiders (Lycosa carolinensis Araneae: Lycosidae) were collected from Yavapai County, AZ and held in individual containers in the Department of Entomology, University of California, Riverside, CA. Whole venom was obtained through an electrical milking technique and held at −80 °C until processed. Prior to fractionation by reversed-phase liquid chromatography, venom was dissolved in 0.1% aqueous trifluoroacetic acid and fractionated with a Brownlee wide-pore C8 column (4.6 × 150 mm) using a linear gradient of aqueous acetonitrile (CH3CN) in constant 0.1% trifluoroacetic acid at a flow rate of 1.0 ml/min. Subsequent purification of biologically active peaks involved variation of organic modifier (acetonitrile orn-propyl alcohol), ion-pairing agent (trifluoroacetic acid or heptafluorobutyric acid), or column (Brownlee C8 or Vydac C4). Synaptosomes were prepared from the whole brains of 14–30-day-old Sprague-Dawley rats as described previously (11Hillyard D.R. Monje V.D. Mintz I.M. Bean B.P. Nadasdi L. Ramachandran J. Miljanich G. Azimi Z.A. McIntosh J.M. Cruz L.J. Imperial J.S. Olivera B.M. Neuron. 1992; 9: 69-77Abstract Full Text PDF PubMed Scopus (440) Google Scholar) and resuspended in “calcium-free” saline (145 mm NaCl, 5 mm KCl, 1.4 mmMgCl2, 1.2 mm NaH2PO4, 10 mm glucose, 20 mm HEPES, adjusted to pH 7.4 with Tris base). In most experiments, 45 μl of resuspended synaptosomes (∼180 μg of membrane proteins) was incubated with 5 μl of test fraction or deionized water (control) for a specified period of time. To this was added 0.8 μm45Ca2+ in 50 μl of either “high potassium” saline (same as calcium-free saline but containing 137 mm KCl and 1.0 mmCaCl2) or “low potassium” saline (similar to calcium-free saline but containing 1.0 mmCaCl2). Following a 3-s incubation, the reaction was stopped by addition of 200 μl of termination solution (30 mm EGTA, 120 mm NaCl, and 5 mm KCl, adjusted to pH 7.6 with Tris base). Extrasynaptosomal45Ca2+ was removed by rapid filtration with washing buffer (145 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1.4 mm MgCl2, and 20 mm HEPES at pH 7.4) using a Skatron cell harvester. Filters were immersed in 4 ml of Beckman Ready Safe scintillation fluid and subjected to scintillation counting. All analyses were performed at the Biotechnology Instrumentation Facility, University of California, Riverside. Matrix-assisted laser desorption time-of-flight mass analyses were performed with a Finnigan Lasermat instrument. Amino acid composition and sequencing analyses were performed as described previously (12Adams M.E. Bindokas V.P. Hasegawa L. Venema V.J. J. Biol. Chem. 1990; 265: 861-867Abstract Full Text PDF PubMed Google Scholar). Synthetic lycotoxin I was prepared by the Sussex Center for Neuroscience, University of Sussex, Sussex, United Kingdom, on an Applied Biosystems Model 432A Synergy automated peptide synthesizer. The peptide was prepared by solid-phase synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) N-terminal protection of amino acids, and amino acids were activated to form an active ester by 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Peptide cleavage from the resin and side chain deprotection were achieved using trifluoroacetic acid and the scavengers thioenisole and ethanedithiol (100 μl each). Cleavage was conducted for 3 h at room temperature, followed by extraction of the peptide using methyl-t-butyl ether to separate scavengers and reaction by-products. The peptide was lyophilized and purified by preparative RPLC 1The abbreviations used are: RPLC, reversed-phase liquid chromatography; PBS, phosphate-buffered saline; EJP, excitatory junctional potential.using an Aquapore Octyl Prep 10 cartridge column (10 × 250 mm) at 5 ml/min with a gradient of 0–60% solvent B (solvent A = 5% CH3CN and 0.1% trifluoroacetic acid; solvent B = 60% CH3CN and 0.08% trifluoroacetic acid). Magainin B (13Chen H.C. Brown J.H. Morell J.L. Huang C.M. FEBS Lett. 1988; 236: 462-466Crossref PubMed Scopus (270) Google Scholar), a synthetic peptide analog related to magainin 2 isolated from Xenopus laevis(14Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (2020) Google Scholar), was a generous gift from Dr. Hao-Chia Chen (National Institutes of Health, Bethesda, MD). Escherichia coli D31 (CGSC 5165) was kindly provided by Dr. B. J. Bachmann (E. coliGenetic Stock Center, Department of Biology, Yale University). E. coli DH5 was from Dr. F. M. Sladek (Department of Entomology, University of California, Riverside). Both yeasts Candida glabrata (ATCC 2001) and Candida albicans (clinical isolate) were from Dr. R. Mehra (Department of Entomology, University of California, Riverside). Lycotoxin I, lycotoxin II, and magainin B were tested for antimicrobial activity against one Gram-positive bacteria (Bacillus thuringiensis subsp. israelensis), two Gram-negative bacteria (E. coli D31 and DH5), and two fungi (C. glabrata ATCC 2001 and C. albicans) by plate growth inhibition assay on thin agar as described below. The E. coli strains and B. thuringiensis were grown in liquid LB broth (1% Bacto-Tryptone, 0.5% Bacto-yeast extract, and 1% NaCl in H2O) (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) to exponential phase with anA 600 of 0.8, representing 109colony-forming units/ml. Bacteria were added to warm (∼45 °C) 0.7% agar in LB broth at a ratio of 1 μl of bacteria/ml of medium. Then, 8 or 35 ml of the bacteria-agarose/LB mixture was poured over the bottom of a 1.5% agarose/LB plate in a 100 × 20- or 150 × 20-mm dish. 5 μl of magainin B or lycotoxins of various concentrations was then applied to the solidified plate surface as discrete drops. After incubation at 37 °C overnight, the effects of magainin B and lycotoxins were recorded as the clear spots in the bacterial lawn. The antimicrobial activities of lycotoxins and magainin B on E. coli D31 in liquid culture were determined in 96-well plates in a final volume of 100 μl as follows. 10 μl of different concentrations of peptides was added to 90 μl of LB broth containing the inoculate of E. coli D31 adjusted to 105 to 106 colony-forming units/ml. After 7 h of incubation at 37 °C, inhibition of growth was determined by measuring the absorbance at 570 nm. Each minimal inhibitory concentration was determined from two independent experiments performed in duplicate. C. albicans and C. glabrata were grown overnight in YTD medium (1% yeast extract, 2% Tryptone, and 2% glucose) at 37 °C, giving an A 600 of ∼0.9–1.0 (representing 107 colony-forming units/ml). 100 μl of the yeast was diluted in 10 ml of sterilized μl of diluted yeast was on 150 × 20-mm plate of YTD medium. 5 μl of magainin B or lycotoxins of various concentrations was then applied to the solidified plate as discrete drops. After incubation at 37 °C overnight, the effects of magainin B and lycotoxins were recorded as the clear spots in the yeast lawn. activity of both lycotoxin I and magainin B was with from in mm and 150 pH by for 5 at × were then incubated at room for 1 h in deionized water in or with synthetic lycotoxin I or magainin B at various concentrations in The were at × for 3 The was from the and its absorbance was at 570 nm. hemolysis and hemolysis were determined following of the of in and Lycotoxin I was on the of using the described previously V.P. Venema V.J. M.E. J. PubMed Scopus Google Scholar). In the of spider venoms for we that venom of the wolf spider L. carolinensis ion in we this effect from of chemical and electrical gradients across from To characterize this activity, we fractionated the venom by reversed-phase liquid chromatography using the described Two were identified and lycotoxins I and 1 Each lycotoxin was purified in to homogeneity and assays used to the purification of the lycotoxins are shown in of synaptosomes by the concentration by to of was following to lycotoxin I (10 or lycotoxin (10 Both lycotoxins (10 in the of that the lycotoxins with the of synaptosomes to amino acid composition of lycotoxin the shown in the presence of was from of lycotoxins not Amino acid composition to the concentrations of lycotoxins in the venom to be mass laser desorption mass of for lycotoxin I and for lycotoxin II. on mass for the lycotoxins were to amino acid acid composition of lycotoxins I and in are from Edman presence of was on presence of was on The in are from Edman The presence of was on in a new The primary structures of the lycotoxins were determined by automated Edman The amino acid of lycotoxin I was identified as IWLTALKFLGKHAAKHLAKQQLSKL-NH2 The of lycotoxin was determined to be KIKWFKTMKSIAKFIAKEQMKKHLGGE-OH The are in with the from amino acid composition analyses The of lycotoxin I was by mass The mass of the peptide was = is to the mass of predicted for the that predicted for the acid is of lycotoxin = with the predicted of for the acid form and for the this we that lycotoxin as the of lycotoxin was for and was for and in a new of lycotoxin was for and was for and in a new To the chemical of lycotoxin I and to for experiments, the peptide was prepared of lycotoxin I and synthetic lycotoxin I a on reversed-phase liquid chromatography 1 of this the presence of both as the was to the of the two that lycotoxin I and synthetic lycotoxin I have RPLC and synthetic peptides were and to be in their to in synaptosomes to the showed that synthetic lycotoxin I an of as with lycotoxin I with an of that synthetic lycotoxin I is and biologically from the The primary structures of lycotoxins are by or A search in the of peptides a a peptide from the of the J. 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