From the Environmental Toxicology Graduate Program and the
Department of Entomology, University of California,
Riverside, California 92521
Two peptide toxins with antimicrobial activity,
lycotoxins I and II, were identified from venom of the wolf spider
Lycosa 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
-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.
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INTRODUCTION |
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
(1-3) and exocytosis proteins (4, 5). Alternatively, many spider
venoms cause cell membrane disruption and consequent tissue necrosis
through enzymatic actions (6-8).
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 (9), and
relatively little is known with regard to the chemical composition of
L. carolinensis venom. Previous studies involving related
species indicate the presence of neuroactive constituents (10).
Here we characterize two peptide toxins from L. carolinensis
venom 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.
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EXPERIMENTAL PROCEDURES |
Toxin Purification--
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 or
n-propyl alcohol), ion-pairing agent (trifluoroacetic acid
or heptafluorobutyric acid), or column (Brownlee C8 or
Vydac C4).
Preparation of Synaptosomes--
Synaptosomes were prepared from
the whole brains of 14-30-day-old Sprague-Dawley rats as described
previously (11) and resuspended in "calcium-free" saline (145 mM NaCl, 5 mM KCl, 1.4 mM
MgCl2, 1.2 mM NaH2PO4,
10 mM glucose, 20 mM HEPES, adjusted to pH 7.4 with Tris base).
Synaptosomal 45Ca2+ Measurements--
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 µM 45Ca2+ in 50 µl of
either "high potassium" saline (same as calcium-free saline but
containing 137 mM KCl and 1.0 mM
CaCl2) or "low potassium" saline (similar to
calcium-free saline but containing 1.0 mM
CaCl2). 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). Extrasynaptosomal 45Ca2+ 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.
Mass Spectrometry, Amino Acid Composition, and Sequencing
Analyses--
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
(12).
Peptide Synthesis--
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 RPLC1
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).
Antimicrobial Assays--
Magainin B (13), a synthetic peptide
analog related to magainin 2 isolated from Xenopus laevis
(14), 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. coli
Genetic 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) (15) to exponential phase with an
A600 of 0.8, representing 109
colony-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 A600 of ~0.9-1.0
(representing 107 colony-forming units/ml). 100 µl of the
yeast was diluted in 10 ml of sterilized water. 500 µl of diluted
yeast was plated evenly on each 150 × 20-mm agarose 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.
Hemolysis Assay--
Hemolytic activity of both lycotoxin I and
magainin B was assayed with heparinized red blood cells from rabbit
(Oryctolagus cuniculus) rinsed three times in PBS (50 mM NaH2PO4 and 150 mM NaCl, pH 7.0) by centrifugation for 5 min at 4310 × g.
Red blood cells were then incubated at room temperature for 1 h in
deionized water (positive control), in PBS (blank), or with synthetic
lycotoxin I or magainin B at various concentrations (6.25-200
µM) in PBS. The samples were centrifuged at 11,640 × g for 3 min. The supernatant was separated from the
pellet, and its absorbance was measured at 570 nm. Zero hemolysis
(blank) and 100% hemolysis controls were determined following
centrifugation of the centrifugates of red blood cells suspended in PBS
and H2O, respectively.
Neuromuscular Pharmacology--
Lycotoxin I was assayed on the
body wall muscles of prepupal house flies (Musca domestica,
NAIDM strain) using the intracellular electrophysiological recording
methods described previously (16).
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RESULTS |
Isolation of Lycotoxins--
In the process of screening spider
venoms for calcium channel antagonism, we found that venom of the wolf
spider L. carolinensis (1:1000 dilution) disrupts
45Ca2+ ion flux in depolarized rat brain
synaptosomes. As we demonstrate below, this effect results from
dissipation of chemical and electrical gradients across membranes
rather than from calcium channel antagonism. To further characterize
this biological activity, we fractionated the venom by reversed-phase
liquid chromatography using the protocols described under
"Experimental Procedures." Two fractions were identified and named
lycotoxins I and II (Fig. 1A).
Each lycotoxin was purified in subsequent steps to homogeneity (Fig. 1,
B and C).

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Fig. 1.
Isolation of lycotoxins from venom of the
wolf spider L. carolinensis. A, whole venom (25 µl) was fractionated on a Brownlee C8 column (300 Å;
4.6 × 250 mm) using a linear gradient of acetonitrile/water
(0.5%/min) in constant 0.1% trifluoroacetic acid. Elution of
lycotoxins I and II is indicated by arrows. B, shown is the elution profile of lycotoxin I using a linear gradient of
n-propyl alcohol/water (0.33%/min) in constant 0.5%
trifluoroacetic acid (same column as described for A).
C, shown is the elution profile of 7.5 nmol of native
lycotoxin I using a Vydac C4 column (300 Å; 4.6 × 250 mm) and an acetonitrile/water gradient (0.25%/min) in constant
0.1% heptafluorobutyric acid. D, shown is the coelution of
native lycotoxin I (4 nmol) and synthetic lycotoxin I (3 nmol) using
the same conditions as described for C. mAU,
milli-absorbance units.
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Calcium flux assays used to follow the purification of the lycotoxins
are shown in Fig. 2. Depolarization of
rat synaptosomes by elevating the external potassium concentration
increases synaptosomal 45Ca2+ levels by almost
2-4-fold, due to opening of voltage-dependent calcium
channels. Depolarization-induced 45Ca2+ uptake
was completely inhibited following pre-exposure to lycotoxin I (10 µM) or lycotoxin II (10 µM) (Fig. 2). Both
lycotoxins (10 µM) also depleted basal
45Ca2+ levels in the absence of potassium
depolarization (Fig. 2). Taken together, these results suggest that the
lycotoxins interfere with the ability of synaptosomes to sequester
Ca2+.

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Fig. 2.
Effects of lycotoxins I and II on
synaptosomal 45Ca2+ levels under depolarized or
resting conditions. Synaptosomes were exposed to 10 µM lycotoxin I (Lyc I) or II (Lyc
II) for 30 min prior to simultaneous addition of
45Ca2+ and either high or low potassium.
Lycotoxins were omitted in respective control experiments. Histogram
values are the average of three experiments, and error bars
are S.E. Further details are given under "Experimental Procedures."
White bars, depolarized conditions; shaded bars,
resting conditions.
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Amino Acid and Mass Analyses--
Quantitative amino acid
composition analysis of each lycotoxin gave the results shown in Table
I; the presence of tryptophan was deduced
from on-line UV absorption spectra of lycotoxins (data not shown).
Amino acid composition analysis allowed us to determine the
concentrations of lycotoxins in the crude venom to be ~5
mM. Preliminary mass analysis (matrix-assisted laser
desorption time-of-flight) gave molecular mass values of 2843 for
lycotoxin I and 3204 for lycotoxin II. Based on molecular mass values
for each toxin, the lycotoxins were estimated to contain ~25-28
amino acid residues, including tryptophan.
Primary Structures of Lycotoxins--
The primary structures of
the lycotoxins were determined by automated Edman degradation. The
amino acid sequence of lycotoxin I was identified as
IWLTALKFLGKHAAKHLAKQQLSKL-NH2 (Table
II). The sequence of lycotoxin II was
determined to be KIKWFKTMKSIAKFIAKEQMKKHLGGE-OH (Table
III). The deduced sequences are in good
agreement with the results from amino acid composition analyses (Table
I). The C-terminal amidation of lycotoxin I was assigned by high
resolution electrospray mass spectrometry. The mass of the native
peptide was [MH]+ = 2843.4, which is virtually identical
to the mass of 2843.5 predicted for the C-terminal amidated peptide,
whereas that predicted for the free acid is 2844.5. Mass analysis of
lycotoxin II yielded [MH]+ = 3206.0, compared with the
predicted values of 3205.8 for the free acid form and 3204.8 for the
amidated form. From this comparison, we concluded that lycotoxin II
occurs as the free acid.
To confirm the chemical identity of lycotoxin I and to provide
sufficient quantities for further biological experiments, the peptide
was prepared synthetically. Co-injection of native lycotoxin I (4 nmol)
and synthetic lycotoxin I (3 nmol) gave a single peak on reversed-phase
liquid chromatography (Fig. 1D); integration of this peak
indicated the presence of both substances as the peak area was
proportional to the total amount of the two materials tested. This
confirmed that native lycotoxin I and synthetic lycotoxin I have
identical RPLC elution profiles. Additionally, native and synthetic
peptides were compared and found to be indistinguishable in their
ability to deplete 45Ca2+ levels in rat
synaptosomes (Fig. 3). Logistic curves
fitted to the data showed that synthetic lycotoxin I had an
EC50 of 5.0 ± 0.13 µM as compared with
native lycotoxin I with an EC50 of 4.5 ± 0.14 µM. Taken together, these results show that synthetic lycotoxin I is chemically and biologically indistinguishable from the
native peptide.

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Fig. 3.
Concentration-dependent reduction
of rat brain synaptosomal 45Ca2+ levels by
native lycotoxin I and synthetic lycotoxin I. Synaptosomes were
preloaded with 45Ca2+ as described in the
legend to Fig. 2 and then incubated with different concentrations of
toxin for 30 min. Intrasynaptosomal 45Ca2+
levels were determined as described under "Experimental
Procedures." Each point is the average of four experiments. ,
native lycotoxin I; , synthetic lycotoxin I.
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Amphipathic
-Helical Character of Lycotoxins--
The primary
structures of lycotoxins are characterized by lysine repeats occurring
every fourth or fifth position. A GenBankTM search resulted in the
identification of several peptides showing a similar lysine motif,
including adenoregulin, a peptide from the skin secretion of the frog
Phyllomedusa bicolor (17), and dermaseptins and magainins
(14, 18) (Fig. 4A). Magainins, dermaseptins, and adenoregulin are all antimicrobial pore-forming peptides with characteristic amphipathic
-helical secondary
structures (14, 17, 18). Interestingly, the predicted secondary
structures of lycotoxins I and II show that the majority of their amino
acids occur in an
-helix conformation according to Chou-Fasman (19) and Garnier-Osguthorpe-Robson (20) principles. When plotted as
-helical wheels, the majority of the hydrophobic and hydrophilic amino acid residues occur on opposite sides of the helix (Fig. 4,
B and C). For lycotoxin I, for instance, six
charged or hydrophilic residues occur on one side of the cylindrical
surface, and eight hydrophobic residues are arranged on the opposite
site. Except for the C-terminal lysine (lysine 24), all positively
charged lysine residues are on the hydrophilic side. For lycotoxin II, six lysines and the C-terminal glutamate occur on the hydrophilic side,
whereas eight hydrophobic residues and two lysines occur on the
predicted hydrophobic side. This suggests that both lycotoxins I and II
may be configured as amphipathic
-helices spanning large portions of
the peptides. In fact, such amphipathic
-helical secondary structure
has been predicted for many other peptides including PGLa, XPF, and
bombinins from frog skin and cecropins from insects (21, 22). The
finding that lycotoxins are significantly conserved with antimicrobial
peptides in both the primary and secondary structures led to
experiments designed to test them for antimicrobial activity.

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Fig. 4.
A, sequence alignment of lycotoxins I
and II with the -helical amphipathic peptides magainin B and
adenoregulin, all of which show a pattern of repeating lysine residues.
B, -helical wheel plot of lycotoxin I showing amphipathic
character. In this conformation, periodic variation in the
hydrophobicity value of the residues along the peptide backbone with a
3.6 residues/cycle period characterizes an -helix (20). The amino
acid sequence of lycotoxin I begins with isoleucine (I) and
proceeds in a clockwise direction. Note that all lysine residues with
the exception of one occur on the "hydrophilic side" (upper
left) of the helix. C, -helical wheel plot of
lycotoxin II. In this plot, the hydrophilic side of the predicted
-helix lies at the lower right. Six positively charged lysine residues occur on the predicted hydrophilic side of the helix,
and two occur on the hydrophobic side.
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Antimicrobial Activity of Lycotoxins--
The lycotoxins show
potent antimicrobial activity against both prokaryotic and eukaryotic
cells in plate growth inhibition assays. Against E. coli
strain DH5 (Fig. 5, rows
A-C), lycotoxin II was most active, showing inhibitory activity
at concentrations as low as 40 µM as compared with 60-80
µM for magainin B and 80-150 µM for
lycotoxin I. The most sensitive to lycotoxin I was a Gram-positive bacterial species, B. thuringiensis israelensis (Fig. 5,
row E), for which the minimal inhibitory concentration of
lycotoxin I was 5 µM (lowest concentration tested); the
minimal inhibitory concentration for magainin B and lycotoxin II was 60 µM (Fig. 5, rows D and F). Against
Gram-negative E. coli strain D31 (23), lycotoxins I and II
were similarly active in the 10-20 µM range (Table
IV). Finally, lycotoxins I and II
inhibited growth of a yeast species, C. albicans, at 40 µM, whereas twice that concentration was required for
observable activity with magainin B (Fig. 5, rows G-I). A
summary of the results from plate growth inhibition assays is shown in
Table IV. In general, the lycotoxins were more active as antimicrobial
agents than magainin B.

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Fig. 5.
Lycotoxins show antimicrobial activity
against both prokaryotic and eukaryotic cells, as demonstrated by
classical zone inhibition assays. White dots represent
inhibition of cell growth at sites of toxin application. Rows
A-C, Gram-negative bacteria (E. coli strain DH5);
rows D-F, Gram-positive bacteria (B. thuringiensis
subsp. israelensis); rows G-I, yeast
(C. glabrata). Magainin B was applied in rows A,
D, and G; lycotoxin I was applied in rows
B, E, and H; and lycotoxin II was applied in
rows C, F, and I. Toxin concentrations
(micromolar) are shown at the top of each column.
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Table IV
Antimicrobial activities of lycotoxin I, lycotoxin II, and magainin B
Toxins of appropriate concentration in water were applied to the top of
agarose plates containing bacteria or yeast. Synthetic lycotoxin I,
native lycotoxin II, and synthetic magainin B were used in the assays.
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When tested in a liquid growth inhibition assay (Fig.
6), the antimicrobial actions of
lycotoxin I, lycotoxin II, and magainin B appeared very abruptly at
submicromolar concentrations (~0.6-0.7 µM). When
applied jointly, the effects of lycotoxins I and II were additive,
suggesting that they have little or no synergistic action. The slope
factors for these inhibition curves were in excess of 25; the steep
relationship between concentration and lytic activity indicates a phase
change in the membrane that most likely involves a high degree of
cooperativity between toxin molecules and is characteristic of peptides
for which aggregation confers antimicrobial activity (24).

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Fig. 6.
Antimicrobial activity of lycotoxin I,
lycotoxin II, and magainin B using an E. coli strain D31
suspension assay. The combined action of lycotoxins I and II is
shown ( ). Inhibition by each toxin was determined after a 7-h
incubation. The control condition involved addition of toxin-free LB
broth.
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Lysis of Erythrocytes--
Pore-forming peptides such as the
magainins lyse erythrocytes at high micromolar concentrations (13).
Given the sequence similarities between the lycotoxins and magainins,
we compared lycotoxin I and magainin B for the ability to lyse rabbit
erythrocytes (Fig. 7). Neither lycotoxin
I nor magainin B showed detectable hemolytic activity at concentrations
below 30 µM. However, both peptides showed significant
hemolytic activity at concentrations above 100 µM, with
lycotoxin I being more active. For instance, at 200 µM,
55% hemolysis was observed for lycotoxin I
((Alycotoxin/AH2O) × 100%) and 35% for magainin B. These results provide further evidence for the pore-forming activity of lycotoxin I.

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Fig. 7.
Hemolytic activity of lycotoxin I compared
with that of magainin B. Release of hemoglobin was determined by
measuring the absorbance at 570 nm. Rabbit erythrocytes were incubated
with increasing concentrations of lycotoxin I ( ) or magainin B ( ) dissolved in PBS for 1 h. The positive control for cell lysis was
determined by addition of water ( ). PBS was used as a blank for the
absorbance measurement.
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Lycotoxin Dissipates Ion Gradients--
To further characterize
the effects of lycotoxins on ion gradients, we performed several
experiments using synaptosomes preloaded with
45Ca2+ (Fig. 8).
Synaptosomes (~180 µg of membrane protein) accumulated a maximum of
65 pmol of 45Ca2+ within 10 min (Fig. 8,
Treatments 1 and 3). Note that Treatment 2, which involved exposure to 45Ca2+ and
cobalt simultaneously, blocked 45Ca2+
accumulation, whereas addition of cobalt after a 10-min delay (Treatment 3) permitted accumulation to the control level.
Cobalt is known to antagonize both inward and outward
45Ca2+ movements due to inhibition of calcium
channels and the Na+-Ca2+ exchanger (25).

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Fig. 8.
Lycotoxin I causes
45Ca2+ efflux from synaptosomes. A,
schematic diagram outlining experiment protocols; B, data.
20 µl of freshly prepared synaptosomes (4 mg/ml) was used for each
treatment. For each treatment, 0.8 µM
45Ca2+ was added in 25 µl of calcium-free
buffer at time 0. In Treatments 2, 3, and
6, Co2+ was added in 5 µl of water at the
times shown to achieve a final concentration of 5 mM. In
Treatments 4-6, lycotoxin I was added in 5 µl of water to
achieve a final concentration of 5 µM. At 25 min, 200 µl of termination solution was added, and the level of
intrasynaptosomal 45Ca2+ was determined by
rapid filtration as described under "Experimental Procedures." The
results are the average of five independent experiments.
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Addition of synthetic lycotoxin I at time 0 (Fig. 8, Treatment
4) or after a 15-min preloading period (Treatment 5)
reduced control 45Ca2+ accumulation by 80%.
The result of Treatment 4 suggests that the toxin prevents
45Ca2+ sequestration, whereas the result of
Treatment 5 indicates that the toxin promotes
45Ca2+ efflux from preloaded synaptosomes. If
preloaded synaptosomes were treated sequentially with cobalt followed
by lycotoxin I, the control response was reduced 50%. Taken together,
these results suggest that lycotoxin I dissipates
45Ca2+ gradients in preloaded synaptosomes.
Lycotoxin I Dissipates Membrane Electrochemical
Potential--
Since insects are the natural prey targeted by wolf
spiders, we tested the effects of lycotoxin I on insect musculature.
Specifically, we examined the effects of the toxin on muscle membrane
potentials. A pore-forming activity of lycotoxin would be manifested as
a diminution of the resting membrane potential. Intracellular
recordings were made from body wall muscles 6A and 7A of larval third
instar house flies (M. domestica). The resting potential
(Em) and amplitude of the neurally evoked excitatory
junctional potential (EJP) were recorded and plotted every 4 s
(Fig. 9). Brief exposure to synthetic
lycotoxin I (1 µM) caused a slight but reversible depolarization of the Em and a corresponding
decrease in the amplitude of the EJP. When 3 µM lycotoxin
I was applied, the Em depolarized from
60 to 0 mV.
At the same time, the EJP was completely blocked. These results show
that lycotoxin I dissipates the muscle cell resting potential in this
preparation.

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Fig. 9.
Lycotoxin I dissipates the membrane potential
of house fly body wall muscle cells. The lower trace
shows the muscle membrane potential (Em) during the
course of the experiment, and the corresponding EJP amplitudes are
plotted in the upper trace. EJPs were evoked by stimulation
of the motor nerve every 4 s. Lycotoxin I was added momentarily to
the bathing medium at 14 min (final concentration, 1 µM)
and again at 29 min (final concentration, 3 µM). The
black bars show the duration of lycotoxin I in the bath at
each application. Note that the Em is depolarized
slightly during the first application and is completely lost in a
reversible manner during the 3 µM application. The
transient reduction of the EJP amplitude results from depolarization of Em. The experiment was performed by Dr. Daewoo
Lee.
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DISCUSSION |
We have identified and characterized the lycotoxins, two
broad-spectrum antimicrobial peptides that occur in venom of the wolf
spider L. carolinensis. To our knowledge, this is the first report documenting the occurrence of antimicrobial peptides in spider
venom. The lycotoxins occur at concentrations of 1-5 mM in
whole L. carolinensis venom, which is in the range
previously reported for agatoxins occurring in funnel web spider
(Agelenopsis aperta) venom (12, 26-28). Such high
concentrations suggest that the lycotoxins play an important role in
the biochemical strategy used by the spider to capture insect prey. We
observed a complete loss of cell membrane potential and block of
neuromuscular transmission in insect body wall muscles by lycotoxin I
(3 µM). Furthermore, the lycotoxins (2-20
µM) caused efflux of calcium ions from rat brain
synaptosomes. These results indicate that the lycotoxins dissipate ion
and voltage gradients across the membranes of excitable cells and
likely contribute to paralysis of envenomated prey.
The utility of the lycotoxins in prey capture is complemented by their
antimicrobial activity, which may serve as a defense against infectious
organisms arising from prey ingestion. Precedent for this concept can
be found in reports of magainins and defensins expressed by epithelial
cells in gastric and intestinal mucosa (29-31), where they may have
multiple functions, including defense against microbial infection and
regulation of symbiotic gut flora (32-35). The mode of action of the
lycotoxins therefore may serve two functions for the wolf spider: as a
paralytic agent used for prey capture as well as a defense against
infectious microbes arising from prey ingestion.
Lycotoxins I and II are cationic peptides containing 25 and 27 amino
acids, respectively. They share 52% sequence similarity with each
other. Theoretical predictions of their secondary structures suggest
the formation of a cationic amphipathic
-helix in which lysine
residues are clustered on the face of the cylindrical surface. This
type of secondary structure is characteristic of magainins, dermaseptins, and adenoregulin. Indeed, lycotoxin I shows 60% sequence
similarity to magainin B and 48% similarity to adenoregulin. Lycotoxin
II shows 45% similarity to magainin B and 48% similarity to
adenoregulin. The antimicrobial actions of the lycotoxins are also
consistent with this group of amphipathic
-helical peptides, which
also includes PGLa, CPF, and XPF (36, 37).
Natural antimicrobial peptides with amphipathic
-helical structures
(38) have been shown to disrupt biological membranes through pore
formation (39-45) or, in some cases, through destabilization of
membrane phospholipid packing (46). These peptides form channels by
self-aggregation of peptide monomers, whereby hydrophilic residues on
one side of the helix face inward and hydrophobic residues on the
opposite side of the helix interact with fatty acid side chains of the
lipid bilayer.
At least four lines of evidence presented in this paper support the
hypothesis that lycotoxins behave as pore-forming peptides. 1) They
inhibit the growth of bacteria at a characteristic critical concentration, an indicator of self-aggregation of the peptide; 2)
cause 45Ca2+ efflux from rat brain
synaptosomes; 3) induce hemolysis of erythrocytes; and 4) dissipate the
electrochemical potential across muscle membrane. Experiments in which
lycotoxins showed antimicrobial activity were conducted in parallel
with magainin B, which showed similar potency in some of the assays. Of
particular interest are the data on lysis of E. coli strain
D31, in which both lycotoxins I and II and magainin B showed extremely
steep concentration response curves (Fig. 6). In contrast, these agents
lysed red blood cells at much higher concentrations with parallel but
far less steep concentration curves (Fig. 7). These data indicate that
the lycotoxins and magainin B operate according to similar but probably
distinct mechanisms in each system. Finally, the amino acid sequences
of lycotoxin I and magainin B are quite similar, both being polar basic
peptides with
-helical amphipathic character. Since magainin B
belongs to a well established family of pore-forming peptides (41, 43),
we believe that the lycotoxins permeabilize cell membranes through
similar mechanisms.
Although the lycotoxins share significant sequence similarity with
magainin B, different specificities were observed for each peptide.
Lycotoxin I was more effective against the Gram-positive bacteria
B. thuringiensis and yeast than magainin B. Magainin B (10 µM) also showed no effect on synaptosomal
Ca2+ efflux (data not shown), whereas both lycotoxins
caused 45Ca2+ efflux from synaptosomes.
The lycotoxins define a new functional subclass of spider venom toxins
based on their amino acid sequences and antimicrobial activity. As with
the magainins, we believe that the lycotoxins have the potential to
stimulate development of new useful therapeutic agents (47, 48). The
rapid development of antibiotic-resistant bacteria points to the
ongoing need for novel antimicrobial agents (38). Our findings suggest
that spider venoms represent a potentially widespread source of novel
antimicrobial agents that may have important medical implications.
We thank Drs. Rajesh Mehra, Frances Sladek,
and B. J. Bachmann for kindly providing microorganisms; Dr.
Hao-Chia Chen for magainin B; Dr. Daewoo Lee for performing the
electrophysiology experiments; Kathy Schegg for help and advice with
amino acid composition and protein sequencing analyses; and Tom
Prentice for expertise in obtaining wolf spider venom. We are also
grateful to Dr. Guoqiang Jiang for help in the antimicrobial inhibition assays and Dr. Janette Mills for valuable discussions during the preparation of this manuscript.