From the Center for Marine Biotechnology and
Biomedicine, Scripps Institution of Oceanography, University of
California, San Diego, La Jolla, California 92093-0204 and the
Departments of ¶ Medicine and
Pediatrics, and the
** Molecular Biology Institute, UCLA,
Los Angeles, California 90095
Received for publication, November 6, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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Plicatamide
(Phe-Phe-His-Leu-His-Phe-His-dc Phe-Phe-His-Leu-His-Phe-His-dc Peptide Purification
Native 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 Synthesis
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 (2, 3). Briefly, the synthetic peptides (1 mg/ml, final
concentration) were dissolved in 10 ml of 20 mM borate, 0.1 M 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 6 N 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.
Antimicrobial Assays
Radial Diffusion Assays--
The assay has been described
elsewhere (4). Our Gram-positive test organisms were
Staphylococcus aureus 930918-3, MRSA ATCC 33591, a
methicillin-resistant S. aureus strain, and Listeria monocytogenes, strain EGD. In some experiments we also tested Escherichia 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 mM
phosphate 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 mM NaCl. 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 log10
of 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).
Colony Counting Assays--
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.
Broth Microdilution Assays--
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 (5), 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.
Potassium Release
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
(6).
Oxygen Consumption
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).
Cytotoxic and Hemolytic Activity
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, where
Aexper and Acontrol
signify 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.
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-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Eq. 1)
Electron Microscopy
For transmission electron microscopy, 5 × 108 bacterial 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.
Planar Lipid Bilayers
Solvent-containing phospholipid bilayer membranes were formed by
placing a small bubble of 15 mg/ml lipid solution in
n-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 (7). 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. The
cis-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.
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RESULTS |
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Antimicrobial Activity of Plicatamide--
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. coli
and 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.
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Composition of Native Plicatamide Preparations--
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.
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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).
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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 mM
Mg2+ impair it (data not shown).
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Effect on Bacterial Membrane Integrity--
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).
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Model Membrane Bilayers--
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 (<10
picosiemens) conductance. At pH 7.4, plicatamide concentrations below 5 µg/ml caused short spikes of increased conductance, whereas concentrations between 5 and 10 µg/ml sometimes induced substantial conductivity (Fig. 6a), with
an essentially linear integral current-voltage response (Fig.
6b). Plicatamide concentrations above 10 µg/ml typically
increased conductance very quickly before destroying the membrane. When
these various concentrations of plicatamide were added at pH 5.5, no
increased conductivity resulted (data not shown).
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The plicatamide-modified membranes were cation-selective, manifesting a
current reversal potential of +15.5 ± 3.5 mV for a 10-fold KCl
gradient. They showed relatively little selectivity for potassium
versus sodium (2.1 ± 1 mV for bi-ionic system at 100 mM, and 10.7 ± 3.2 mV for 100 mM KCl, 1 M NaCl system). Although adding 1-10 mM
CaCl2 had no effect on plicatamide-induced currents, adding
1 mM ZnCl2 blocked current flow by up to 75%
(data not shown). Because we have found that plicatamide binds
zinc,2 we attribute the
inhibitory effect of this cation to its interaction with plicatamide,
rather than to nonspecific stabilization of the membrane. It is
noteworthy that ZnCl2 blocks pore formation by two
histidine-rich polypeptides: aerolysin from Aeromonas
hydrophila (8, 9) and a histidine-rich analogue of staphylococcal
-hemolysin (10).
Oxygen Consumption--
Exposing MRSA to plicatamide or PL-101
quickly decreased their consumption of oxygen by 87.1%, from a basal
rate of 5.1 to 0.66 nmols/min/108 CFU, within 60 s
after 10 µg/ml native plicatamide was added (Fig.
7). Synthetic PL-101 was almost as
effective, reducing O2 consumption by 81.8% to 0.93 nmols/min/108 CFU. Untreated control organisms continued to
consume O2 at the basal rate until the chamber became
anaerobic.
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Microbroth Dilution Assays--
We also performed conventional,
National Committee for Clinical Laboratory Standards (NCCLS)
microbroth dilution assays to determine the susceptibility
of E. coli ML-35p, P. aeruginosa MR3007, S. aureus 930918-3, and L. monocytogenes EGD to native plicatamide and PL-101. In contrast to its prominent antimicrobial effects in our radial diffusion and colony count experiments, the MIC
of plicatamide exceeded 100 µg/ml for each of the aforementioned organisms. Because microbroth dilution assays are widely considered to
represent "gold standards" in testing antimicrobials, we decided to
investigate the cause of this apparent discrepancy. Initially, we
suspected that the Mueller-Hinton broth used in National
Committee for Clinical Laboratory Standards-type assays might not
support plicatamide mediated staphylocidal activity. However, when we exposed mid-logarithmic or stationary phase S. aureus to 2 or 5 µg/ml of plicatamide in Mueller-Hinton broth, the colony counts fell by >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.
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Effects on Bacterial Ultrastructure--
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.
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Hemolytic and Cytotoxic Properties--
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).
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DISCUSSION |
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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 (11-15). A few smaller antimicrobial peptides with
11-13 residues have been described. These include the bactenecin
dodecapeptides of bovine or ovine neutrophils (16, 17), bovine
indolicidin (18, 19), and tigerinins, antimicrobial peptides isolated
from the skin secretions of a frog, Rana tigerina (20).
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.
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To our knowledge, only two smaller antimicrobial peptides
have been found in animals: 5-S-GAD, and halocyamine A. N--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)
(21). 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 (22).
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) (23, 24).
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 bacteria Achromobacter aquamarinus and Pseudomonas perfectomarinus (25). 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 dcDOPA might be to impart
adhesive properties that help retain the peptide at sites of injury or
infection. Byssal threads and plaques, the major adhesive structures of
marine mussels (Mytilus spp.), invariably contain DOPA (26).
If a Lewis base and an oxidase such as polyphenol oxidase are both
present, DOPA can be converted to a DOPA quinone, whose spontaneous
tautomerization forms
,
-dehydro-DOPA (27, 28).
Tunicates are protochordates-invertebrates that belong to the phylum chordata. Although the functional biochemistry of tunicate hemocytes has received relatively little attention, much is known about the microbicidal mechanisms of mammalian white blood cells, especially polymorphonucleated granulocytes (PMN). Mammalian PMN employ two principal strategies to kill microorganisms. One strategy involves using an array of antimicrobial peptides and proteins (29), and the other depends on the production of oxidants by post-phagocytic metabolism (30). The principal oxidants of human PMN are produced by an NADPH oxidase complex (31, 32) and include H2O2, OH (hydroxyl radical), and "downstream" products such as chloramines and hypochlorous acid formed by interactions between H2O2 and myeloperoxidase (33). In the PMN of rodents and some other mammals, copious amounts of nitric oxide are formed by an inducible nitric-oxide synthase (34).
Although it is not known if tunicate hemocytes have NADPH oxidase or inducible nitric-oxide synthase activity, phenol oxidase is released from the hemocytes of H. roretzi after foreign cells (e.g. yeast) are encountered. H. roretzi phenol oxidase, a metalloenzyme that requires copper ions for full activity (35), was reported to be antibacterial in the presence of DOPA and H. roretzi hemolymph. Because certain hemocytes ("morula cells") of the colonial ascidian Botryllus schlosseri also contain phenoloxidase (36), this enzyme may be widely distributed in tunicates. The possibility that the DOPA moiety of plicatamide endows the molecule with an ability to participate in oxidative microbicidal reactions deserves consideration, especially because its histidines and modified DOPA residue endow plicatamide with the ability to bind transition metals.2
Whereas direct measurements of potassium efflux have seldom been applied to antimicrobial peptides, many investigators have used membrane potential sensitive carbocyanine dyes to follow their effects on bacterial membrane potential. For example, in a recent study of S. aureus and S. epidermidis, Hancock and co-workers (37) compared the kinetics of killing (by colony counts) with that of membrane depolarization. At early time points, when membrane depolarization was incomplete, 90% or more of the bacteria had been killed. These results are relevant to our findings with plicatamide, because membrane potential and intracellular potassium concentrations are related by the Nernst equation: Eeq = (RT/F) ln((Ko)/(Ki)). In this equation, Eeq represents the membrane potential at equilibrium; (Ko)/(Ki) is the ratio of potassium concentrations outside and inside the cell; ln represents natural logarithm; T is the absolute temperature; R is the universal gas constant; and F (the Faraday) is a physical constant. In our studies, the fate of plicatamide-treated bacteria appeared to be determined as soon as the process responsible for their potassium loss began, which should correlate with the onset of the depolarization process.
The ability of plicatamide to induce a massive potassium efflux from staphylococci suggests that it acts on their plasma membrane. The rapidity of its lethal and leakage effects prove that the thick staphylococcal cell wall peptidoglycan is not a barrier to the diffusion of plicatamide. The decreased activity of plicatamide under acidic conditions (pH 5.5), when its net positive charge would be highest, suggests that electrostatic interactions (e.g. with anionic phospholipids of the bacterial membrane) are unlikely to play a major role in its activity. The data reported here show that plicatamide retains its antimicrobial activity in our "high salt" conditions (250 mM). More significantly, at least from the perspective of a tunicate, plicatamide retained full activity in media containing 450 mM NaCl (i.e. the NaCl concentration of seawater) and in seawater itself (data not shown).
The striking morphological changes in plicatamide-treated MRSA somewhat resemble the membrane "blebbing" often shown by Gram-negative bacteria after exposure to antimicrobial peptides (38, 39). However, because Gram-positive bacteria lack an outer membrane and encase their cytoplasmic membrane within a thick cell wall, these changes have a different genesis. We interpret the bleb-like protrusions of plicatamide-treated MRSA as osmotically driven herniations of the plasma membrane through small clefts ("tesserae") in the peptidoglycan fabric of the cell wall. We have seen similar changes with other antimicrobial peptides, especially protegrins (40), and will describe them in detail elsewhere.
Whereas ovine erythrocytes were resistant to lysis by plicatamide (Fig. 12), human erythrocytes were susceptible. Although the reasons for this difference are unclear, very similar findings have been noted with respect to lysis by other antimicrobial peptides (SMAP-29 and protegrin PG-1) (13, 41), bile salts (42), or hypotonic conditions (43). Several factors may contribute to the differential susceptibility of human and sheep erythrocytes to lysis. Among these are their marked difference in size (sheep red cells are considerably smaller) and phospholipid composition (sheep erythrocytes lack phosphatidylcholine) (44, 45). In addition, some strains of sheep have red blood cells whose intracellular ionic composition differs greatly from that of human erythrocytes (46).
From the standpoint of humans, the rapid and potent effects of
plicatamide and PL-101 on staphylococci are also of interest. Infections caused by glycopeptide-resistant staphylococci and enterococci are becoming increasingly common, and additional agents that are effective against VanA strains of enterococci and
"GISA"-type S. aureus are urgently needed (47). Although
plicatamide was not especially cytotoxic and had little hemolytic
activity for sheep erythrocytes, both plicatamide and PL-101 were
extremely hemolytic for human red blood cells. We are
currently trying to design active oligopeptide analogues of plicatamide
with an improved cytotoxicity/hemolysis profile. If successful, these
efforts could lead to practical applications.
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ACKNOWLEDGEMENTS |
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We thank Tung X. Nguyen, Ehsan Fam, Dmitri S. Orlov, and Huiyuan Wu for technical assistance.
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FOOTNOTES |
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* This work was supported in part by awards from the Stein-Oppenheimer Endowment Fund and the National Sea Grant Program of the United States Department of Commerce, National Oceanic Atmospheric Administration Grant R/MP-93 (through the California Sea Grant College Program and the California State Resources Agency).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Preliminary studies were supported by a generous donation from the Kieckhefer Foundation.
To whom correspondence should be addressed: Dept. of Medicine,
UCLA Center for Health Sciences, 10833 LeConte Ave., Los Angeles, CA
90095. Tel.: 310-825-5340; Fax: 310-206-8766; E-mail:
rlehrer@mednet.ucla.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M211332200
2 J. A. Tincu, L. P. Menzel, R. Azimov, J. Sands, T. Hong, A. J. Waring, S. W. Taylor, and R. I. Lehrer, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
plicatamide, Phe-Phe-His-Leu-His-Phe-His-dcDOPA;
dc
DOPA, decarboxy-(E)-
,
-dehydro-3,4-dihydroxyphenylalanine;
DOPA, 3,4-dihydroxyphenylalanine;
MSRA, methicillin-resistant S. aureus;
CFU, colony-forming units;
PBS, phosphate-buffered saline;
FTIR, Fourier transform infrared;
MEC, minimal effective concentration;
MES, 4-morpholineethanesulfonic acid;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
HPLC, high pressure liquid chromatography;
AUC, area under the curve;
5-S-GAD, N-
-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine;
PMN, polymorphonucleated granulocytes.
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