From the Laboratoire d'Ethologie Expérimentale
et Comparée, CNRS ESA 7025, Université Paris 13, avenue JB
Clément, 93430 Villetaneuse, France, § Laboratoire de
Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, Ecole
Supérieure de Physique et de Chimie Industrielles de la Ville de
Paris, 10 rue Vauquelin, 75005 Paris, France,
Laboratoire de
Fermentations et de Bioconversions Industrielles, Ecole Nationale
Supérieure d'Agronomie et des Industries Alimentaires, Institut
National Polytechnique de Lorraine (ENSAIA-INPL), 2 avenue de la
Forêt de Haye, BP 172, 54505 Vandoeuvre-les-Nancy, France,
** Laboratoire de Chimie des Substances Naturelles, CNRS ESA 8041, Museum National d'Histoire Naturelle, 63 rue Buffon, 75005 Paris,
France,
Unité de Biochimie
Cellulaire, CNRS URA 1129, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France, and §§ Laboratoire d'Ecologie
Terrestre, CNRS UMR 5552, Université Toulouse III, 118 route de
Narbonne, 31062 Toulouse Cedex, France
Received for publication, January 10, 2001, and in revised form, February 15, 2001
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ABSTRACT |
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The antimicrobial,
insecticidal, and hemolytic properties of peptides isolated from the
venom of the predatory ant Pachycondyla goeldii, a member
of the subfamily Ponerinae, were investigated. Fifteen novel peptides,
named ponericins, exhibiting antibacterial and insecticidal properties
were purified, and their amino acid sequences were characterized.
According to their primary structure similarities, they can be
classified into three families: ponericin G, W, and L. Ponericins share
high sequence similarities with known peptides: ponericins G with
cecropin-like peptides, ponericins W with gaegurins and melittin, and
ponericins L with dermaseptins. Ten peptides were synthesized for
further analysis. Their antimicrobial activities against Gram-positive
and Gram-negative bacteria strains were analyzed together with their
insecticidal activities against cricket larvae and their hemolytic
activities. Interestingly, within each of the three families, several
peptides present differences in their biological activities. The
comparison of the structural features of ponericins with those of
well-studied peptides suggests that the ponericins may adopt an
amphipathic Social insects have developed a number of defensive systems that
prevent the development of disease within colonies. For example, bee
propolis and royal jelly present antimicrobial properties (1, 2), and
the fecal pellets of termites inhibit the development of fungal
pathogens (3). Within ants, most species possess metapleural glands on
the thorax whose secretions, spread over the ants and throughout the
nest, have a broad spectrum of antimicrobial action (4-7). The
mandibular gland secretions of some army ant species also have a dual
defensive role against both predators and microbial attacks of brood
(8). If these mechanisms control the proliferation of many bacteria and
fungi in the nesting environment, the introduction of pathogens may
also arise from alimentation, especially prey.
Among ants, predators stricto sensu are overrepresented
within the subfamily Ponerinae (9). Most of these species capture almost every encountered prey using their venom (10), which contains
peptides (and proteins) (9, 11, 12). Because these prey are then
brought back to the nest immediately after immobilization, their
potential infection by bacteria, fungi, or viruses may seriously affect
the survival of ant colonies or induce extensive damages because
of the high population density combined with the close genetic
relationship of the individuals.
The antibacterial property of ant venom has only been demonstrated in
the fire ant, Solenopsis invicta, in whose venom alkaloids inhibit the growth of both Gram-positive and Gram-negative bacteria and
presumably act as a brood antibiotic (12, 13). Venoms of the wasp,
Vespa crabro, honey bees, and various snakes contain antimicrobial peptides, but their functions have not been investigated (14-17). Finally, lycotoxins isolated from spider venom are the only
antimicrobial peptides in venom for which a preventive role against
infections arising from prey ingestion has been demonstrated (18).
Here we investigate the possible role of the venom of a predatory ant
species in the prevention of microbial disease. The antimicrobial,
insecticidal, and hemolytic properties of the venom of the arboreal
ponerine ant, Pachycondyla goeldii, were studied. In total,
15 novel peptides, named ponericins, were purified, and their primary
structures were fully characterized through amino acid sequencing and
matrix-assisted laser ionization/desorption time-of-flight mass
spectrometry analyses. According to their amino acid sequences,
ponericins were classified into three families named ponericin G, W,
and L. Ten peptides were synthesized to perform detailed analyses of
their biological activities. The relationships of these peptides with
known antimicrobial peptides are discussed.
Ants and Venom
Whole venom reservoirs were dissected from P. goeldii
ants collected in Petit Saut, French Guiana. After rinsing in water, they were stored at HPLC1
Purification
Whole venom was injected into a C18 reversed-phase
column (5 µm, particle size; 220 × 2.1-mm column; Vydac), and
separations were performed at a flow rate of 200 µl/min. Peptide
elution was monitored at 215 nm. Collected fractions were vacuum-dried
and tested for biological activity. First, the venom was separated into
four fractions with a gradient consisting of 10-80% solvent B (80%
acetonitrile in 0.1% trifluoroacetic acid) for 60 min. Solvent A was
0.1% aqueous trifluoroacetic acid. Second, the venom was further
purified on the same column with a biphasic gradient of 10-25%
solvent B for 10 min and 25-55% solvent B for 75 min to
improve the separation of the peptides contained in the active fraction.
Mass Spectrometry
The purified peptides were analyzed by matrix-assisted laser
ionization/desorption time-of-flight (Voyager Elite; PerSeptive Biosystems, Inc., Framingham, MA) mass spectrometry as described by
Seon et al. (19).
Microsequence Analysis
Peptides were sequenced by automated Edman degradation using a
Procise pulse-liquid protein sequencer (model 494; PerkinElmer Life Sciences).
Carboxypeptidase Y Digestion
About 30-60 pmol of peptide were dried and solubilized in 10 µl of 0.1 M ammonium acetate buffer, pH 5.5. Carboxyl-terminal sequences were determined by digestion with
carboxypeptidase Y and mass spectrometry analysis of the digestion
products as described by Seon et al. (19).
Peptide Synthesis
Ten peptides were synthesized by Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by
Synt:em (Nimes, France). Peptides G1, G3, G4, G6, W1, W4, W5, W6, and
L2 were identical to the isolated ponericins. Due to some problems,
ponericin W3 was synthesized in a modified form compared with the
natural peptide. The corresponding synthetic peptide was called W3-desK
and presented 3 lysine residues at the carboxyl-terminal region,
therefore having one lysine residue less than the natural W3
(i.e. Antimicrobial Assays
Step 1--
Crude venom and an aliquot of each HPLC fraction
were tested for their antibacterial activity against one Gram-negative
strain (Escherichia coli RL65) and one Gram-positive strain
(Staphylococcus aureus 209P). Inhibitory activity was
determined by the agar-well diffusion method. Wells 6 mm in diameter
were filled with 10 µl of the sample solution. After 24 h at
37 °C, inhibition zone diameters were measured by subtracting the
well diameter.
Step 2--
Synthetic peptides were tested against a wide range
of Gram-negative and Gram-positive microbial strains, the yeast and
fungi indicated in Table I. Inhibitory activity was determined by the agar-well diffusion method. A 12-ml sample of nutrient broth containing 1.2% agar (bacteriological agar; Biokar) and supplemented with 1 ml/liter Tween 80 (Merck, Darmstadt, Germany) was inoculated with a
fresh overnight culture of the indicator strain (absorbance at 660 nm = 0.01) and poured into Petri dishes. Wells 5 mm in diameter
were filled with 20 µl of the sample solution. These plates were
placed overnight at 4 °C to allow the diffusion of the inhibitory
agent and incubated for 24 h at 30 °C or 37 °C, depending on
the indicator strain. Antifungal activity was also assessed by the
agar-well diffusion assay. The fungal colonies were inoculated into the
agar close to a well filled with the sample solution, and any deviation
from circular growth of the fungal colonies after 24 h of
incubation was scored as fungal inhibition.
The minimal inhibitory concentration (MIC) of the peptides (G1, G6, W1,
W3-desK, and L2) was determined by liquid and plate growth inhibition
assays on the bacterial strains indicated in Table II. In the plate
growth inhibition assay, the MICs were determined by a critical
dilution assay from an initial sample concentration of 64 µM by 2-fold dilutions in a 5 mM phosphate buffer, pH 6.5. Inhibitory activity determination was performed as
described previously. In the liquid growth inhibition assay, 270 µl
of a mid-logarithmic phase culture of bacteria were added to 30 µl of
peptides. Microbial growth was assessed in microtiter plates by an
increase in A620 after 24 h at 30 °C or
37 °C. The concentrations tested for each peptide were in the range
of 0.125-64 µM. The MIC values are expressed as
intervals between the highest concentration at which bacteria grow and
the lowest concentration that causes 100% growth inhibition (20).
Cecropin B from the moth Hyalophora cecropia, melittin from
honey bee venom, and dermaseptin from the skin of the frog
Phyllomedusa sauvagii (Sigma) were used as control
antibiotic peptides in the plate growth inhibition assay.
Hemolytic Assays
A 12-ml sample of blood agar medium was supplemented with 1 ml
of sheep or horse erythrocytes. Wells 5 mm in diameter were filled with
20 µl of the peptide solution and placed overnight at 4 °C to
allow the diffusion of the hemolytic agent. After 24 h at room
temperature, complete hemolysis and lysis zone diameters were measured.
Insecticidal Assays
Given quantities of each synthetic peptide or a mixture of 5 µg of each of the 10 peptides was solubilized in an insect
physiological saline buffer. These peptide solutions were injected into
groups of 10-15 cricket larvae, Acheta domesticus
(weight = 10 ± 0.5 mg), or P. goeldii workers
(mean weight = 8.93 mg). Controls receiving injections of an
insect saline buffer or a dried solution of acetonitrile and
trifluoroacetic acid redissolved in a saline buffer showed no effect.
The lethal dose corresponding to the mortality of 50% of the treated
larvae after 24 h (LD50) was calculated by probit analysis (21).
Circular Dichroism Spectra
The far-ultraviolet CD spectra were recorded in a CD6
spectropolarimeter (Jobin-Yvon, Longjumeau, France) using a 0.01-cm pathlength cuvette. The four synthetic peptides, ponericins G1, G6, W1,
and L2, were solubilized in milliQ water with and without trifluoroethanol (TFE) at 25% at concentrations of 0.95, 0.87, 0.97, and 0.95 mg/ml, respectively. The CD spectra were acquired between 180 and 260 nm, with a 0.5 nm step, a 2-s integration time, and 2 nm
constant bandpass. Each spectrum was averaged from five successive
scans. The baselines (water and 25% TFE) were acquired independently
under the same conditions and then subtracted from the corresponding
sample spectra. The predicted secondary structure content was deduced
from each spectrum using the Varselec analysis (22).
Isolation of Antibacterial Peptides from the Venom of P. goeldii--
An antibacterial assay of the crude venom of P. goeldii showed strong action against both Gram-positive (S. aureus 209P) and Gram-negative (E. coli RL65) bacterial
strains at a 30 µg (dry weight of venom)/µl concentration. The
venom was separated on a reversed-phase HPLC (Fig.
1a) into four fractions, of
which only fraction C showed an antibacterial activity against the two previous strains. Further fractionation of the peptides eluted in
fraction C demonstrated that 12 of the collected peaks had antibacterial properties (Fig. 1b).
Primary Structure Determination of the Antibacterial
Peptides--
The active peptides present in fraction C were first
analyzed by matrix-assisted laser ionization/desorption time-of-flight mass spectrometry. Peaks containing one or two major components were
further studied. In total, 15 peptides were characterized at the level
of their amino acid sequences using Edman degradation. The amino acid
sequence determination was also possible for peaks containing two
peptides present in different quantities. For some of the analyzed
peptides, carboxypeptidase Y digestions were used to identify or
confirm amidation of the carboxyl-terminal amino acid. The accordance
of the experimental masses with the calculated molecular masses was
used as a control for each sequence determined.
These peptides can be classified into three families according to their
primary structures (Fig. 2). The peptides
were named ponericins G, W, or L according to the first most frequent
amino-terminal amino acid. Within each family, most of the peptides
share a great percentage of sequence similarity with each other (up to
87%, 92%, or 96% for ponericins G, W, or L, respectively). The
sequence similarities were calculated using identical residues and
conservative replacements. Ponericins G6, G7, and W6 present the lowest
sequence identities with the other members of the ponericin G and W
families (from 17.2% to 26.7% for G6 and G7 and from 19.2% to 45.8%
for W6). They were nonetheless classified in the latter families
because they retained some common sequence features with the other
members. To further investigate the antibacterial spectrum and
insecticidal activity of each individual peptide, 10 of these 15 peptides corresponding to representative molecules from each family
were synthesized as indicated under "Materials and Methods."
Activity Spectrum of Synthetic Peptides against Various Microbial
Strains--
Whole venom was active against all the tested microbial
strains except the two fungi (Table I).
Among the 31 bacterial strains, the most sensitive Gram-positive
bacteria were Bacillus stearothermophilus, B. subtilis, B. megaterium, and Lactococcus
lactis ssp. cremoris. Pseudomonas aeruginosa
was the most sensitive Gram-negative bacteria.
The 10 synthetic peptides exhibited four types of antibacterial action
spectra at a concentration of 0.4-0.5 mM. Ponericins G1
and G3 exhibited an action highly comparable to that of crude venom and
were active against all the tested bacteria and the yeast. The second
group corresponds to the peptides W1, W3-desK, W4, W5, and L2. For this
group, the results of the growth inhibition assay against Gram-positive
bacteria were also comparable to those with crude venom, but at the
tested concentration, these peptides were less active against most of
the Gram-negative bacterial strains. Within this group, L2 was the only
one that did not affect the yeast Saccharomyces cerevisiae.
The third group of peptides (G6 and W6) was active against most of the
Gram-positive strains but inactive against the Gram-negative strains,
with the exception of P. aeruginosa and Serratia
marcescens. Finally, ponericin G4 only affected a few of the
tested strains. It should be noted that all of the peptides were
inactive against the fungal strains, as observed for crude venom.
Synthetic peptides G1, G6, W1, W3-desK, and L2 were selected for
further analyses. At least one member of each peptide family presenting
a large activity spectrum was retained, except for G6, which was highly
active against only a few strains. Their MICs were determined in both
liquid and plate growth inhibition assays against Gram-positive and
Gram-negative bacterial strains highly sensitive or affected by most of
the chosen peptides (Table II).
In the liquid growth inhibition assay, G1, W1, and L2 had a marked
activity (MIC < 8 µM) against most of the bacterial
strains, whereas W3-desK and particularly G6 were less effective. In
the plate growth inhibition assay, MICs were at least two times higher, highlighting the problem of peptide diffusion in a solid medium. Nevertheless, the same kind of results were observed, with a marked action of peptide G1 against most of the tested bacteria. Furthermore, the comparison of their MICs in the plate growth inhibition assay with
those of cecropin, melittin, and dermaseptin used as reference peptides
showed that their antimicrobial activity is comparable or even higher
for ponericins.
Hemolytic Activity--
The hemolytic activity of the crude venom
and the 10 synthetic peptides was tested on both horse and sheep
erythrocytes at the same concentration as the antimicrobial activity
spectrum (Table I). When affected, the horse erythrocytes were always the most sensitive. Whole venom and three peptides (W1, W5, and W6)
induced total lysis of both horse and sheep erythrocytes, whereas
W3-desK and W4 were less active and exhibited hemolytic activity only
against horse erythrocytes. The remaining five peptides did not exhibit
any hemolytic activity.
Insecticidal Activity--
The insecticidal properties of P. goeldii venom were only detected in fraction C. All 10 synthetic
peptides exhibited insecticidal properties against A. domesticus (Table III). Four were
highly active against crickets (G1, G3, W3-desK, and W4), with toxicity (LD50) < 130 µg peptide/g animal weight. Among
these four peptides, W3-desK and W4 were the only compounds that
affected P. goeldii workers. However, the level of toxicity
against ants was relatively low, suggesting a possible immunization of
these ants against their own venom.
Quantification of Peptides in the Venom Gland--
The
concentration of each of the 10 peptides varied from 0.33 to 22.69 mM in the venom (Table IV).
The results obtained from the plate growth inhibition assay, in which
the tested concentrations of each compound were between 0.4 and 0.5 mM, showed that most of the bacterial strains were highly
affected. Therefore, it appears that even the less concentrated
antibacterial peptide G1 is sufficient in concentration to exhibit a
marked antibacterial activity in the crude venom.
Circular Dichroism Spectra--
To compare some structural
features of the ponericins with well known antibacterial peptides for
which extensive structural data have been obtained, we analyzed the
far-ultraviolet CD spectra of four synthetic peptides in water and in
25% TFE. The four synthetic peptides exhibited similar spectra in
water as well as in TFE. The spectra obtained for ponericin L2 are
shown as example in Fig. 3. In water, the
spectra are typical of a nonperiodic or disorganized structure (random
coil), with a characteristic minimum around 195-200 nm. The spectra
recorded in the presence of 25% TFE, a solvent decreasing the
dielectric constant of water, revealed the presence of a high helical
content, with a typical maximum at 192 nm and minima at 207 and 222 nm.
The Varselec analysis of the spectra obtained in 25% TFE resulted in a
helical content over 20% for all four peptides considered.
This study constitutes the first report on the isolation and
characterization of antibacterial peptides from ant venom. All 15 peptides, named ponericins and identified from the venom of the
ponerine ant P. goeldii, present new sequences not recorded in data bases. They were classified into three different families according to their primary structure similarities: ponericins G, W, and L.
From the first family of peptides, ponericins G, two patterns of
action can be defined. G1 and G3 have a marked action against all the
microbial strains and are also strong insecticides, whereas G4 and G6
are only active against some Gram-positive bacteria and yeast and
present poor insecticidal properties. Ponericins G share about 60%
sequence similarity with cecropins (calculated using G1; Fig.
4a). Cecropins represent a
family of inducible antimicrobial peptides that have been isolated from
only two insect families, Diptera and Lepidoptera (23). To the best of
our knowledge, the ponericins G represent the first molecules isolated
from another insect family that can be considered as cecropin-like
members. All cecropins, except those isolated from Aedes
mosquitos (24-26), are amidated, whereas this posttranslational
modification does not occur in the ponericins G, with the exception of
G6. Cecropins have a broad spectrum of activity against bacteria and
fungi but do not affect most other eukaryotic cells (23, 27). The
strong insecticidal properties of G1 and G3 imply that these peptides are effective against eukaryotic cells with a certain specificity because they do not exhibit any hemolytic action.
-helical structure in polar environments, such as cell
membranes. In the venom, the estimated peptide concentrations appear to
be compatible with an antibacterial activity in vivo. This
suggests that in the ant colony, the peptides exhibit a defensive role
against microbial pathogens arising from prey introduction
and/or ingestion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C until processed. The venom reservoirs were disrupted by ultrasonic waves in a 30% acetonitrile, 0.2% trifluoroacetic acid solution (10 µl/venom reservoir), and
empty reservoirs and membranes were discarded by centrifugation.
LKKKQ for W3-desK instead of
LKKKKQ for W3).
Because W1 and W5 peptides present the same difference in their
carboxyl-terminal sequence (i.e.
FKKKKQ for W1 and
FKKKQ
for W5) together with other small differences (addition or deletion)
but have similar activity spectra (Table I), the W3-desK peptide was
further analyzed to obtain an idea of the biological activity of W3,
despite their minor difference in the carboxyl terminus.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 1.
a, reversed-phase HPLC of the venom of
P. goeldii. Of the four fractions, only fraction C
(shaded) was active against S. aureus (209P) and
E. coli (RL65). b, reversed-phase HPLC optimized
for fraction C separation. Twelve of the collected peaks (indicated by
the names of the peptides) were active against S. aureus and
E. coli.
View larger version (33K):
[in a new window]
Fig. 2.
Amino acid sequences and molecular masses of
the antibacterial peptides isolated from the venom of P. goeldii. Gaps were introduced to optimize the
alignments. Identical residues and conservative replacements are
indicated. The indicated molecular masses correspond to the masses of
the protonated peptides because they were determined experimentally and
calculated after the complete characterization of their sequence.
Peptides for which digestion with carboxypeptidase Y (CPY)
was performed are indicated.
Antimicrobial and hemolytic action spectra of 10 synthetic peptides
from P. goeldii venom
Antibacterial action of ponericins G1, G6, W1, W3-desK, and L2
Insecticidal action (in µg venom/gram injected individual) of the 10 synthetic peptides on the cricket, Acheta domesticus and ant
workers of P. goeldii
Quantity and concentration of the 10 synthesized peptides present in
one venom reservoir of P. goeldii
View larger version (12K):
[in a new window]
Fig. 3.
Circular dichroism spectra of
synthetic ponericin L2 (0.95 mg/ml) in pure water (dotted
lines) or in 25% trifluoroethanol in water (solid
line) in the far-ultraviolet region (180-260 nm). The
solvent contribution was subtracted from the sample spectrum.
Whereas in water no conformational preference was observed, the
Varselec analysis of the spectrum obtained in 25% TFE resulted in a
helical content of about 39% for ponericin L2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 4.
a, comparison of sequences between
ponericin G1 and three cecropins isolated from Diptera
(Drosophila melanogaster and Aedes aegyptii) and
Lepidoptera (Bombyx mori). b, comparison of
sequences between ponericin W1 and gaegurin 5 and melittin.
c, comparison of sequences between ponericin L1 and
dermaseptins 3 and 5. Gaps were introduced to optimize the alignments.
Identical residues and conservative replacements are indicated.
Asterisks indicate identical residues, two dots
indicate conservative replacements, and one dot indicates
semiconservative replacements.
Most peptides from the second family, ponericins W, share common properties linked to their high sequence similarities. With the exception of W6, which presents the lowest sequence similarities with the other ponericins W, this family is active against Gram-positive and Gram-negative bacteria and yeast and has marked hemolytic and insecticidal activities. This peptide family shares about 70% sequence similarity with gaegurin 5 and melittin (calculated from W1 and W3; Fig. 4b). Gaegurin 5, isolated from the skin of the frog Rana rugosa (28, 29), exhibits a broad spectrum of antimicrobial action against bacteria, fungi, and protozoa but has very little hemolytic action, in contrast to the ponericins W. Melittin is the major toxic component of the venom of honey bees and also has high antimicrobial and hemolytic activities (16, 30).
The ponericin L2 from the third family has only an antibacterial action and is inactive against yeast, erythrocytes, and insects. Together with L1, L2 shares important sequence similarities with dermaseptin 3 and 5 that have been isolated from frog skin and possess a strong antimicrobial action against bacteria, yeast, fungi, and protozoa (Fig. 4c) (29, 31, 32). As observed for dermaseptins, no hemolytic action was found for L2. Moreover, this compound is not a strong insecticide and should thus be considered to have a selective antibacterial role in P. goeldii venom.
Interestingly, when compared with the cecropin B, melittin, and dermaseptin used as references in antimicrobial tests, the ponericins showed a similar or higher level of activity. Their activity is also comparable to those of other antimicrobial peptides (20, 23, 24, 33, 34).
Secondary structure predictions using the consensus prediction (35) at
the Network Protein Sequence analysis web server suggested that
ponericins form a full-length -helix or two
-helices separated by
a bend. The helical wheel representations also indicated distinguishable hydrophobic and hydrophilic domains. This suggests that
the ponericins display a similar linear amphipathic
-helical structure. CD spectra performed in the far-ultraviolet confirmed the
adoption of an
-helical structure in the presence of TFE for each of
the three ponericin families (G, W, and L) but also indicated that the
ponericins were unstructured under aqueous conditions. These
observations are consistent with more extensive structural data
obtained for the homologous peptides cecropins, gaegurins, melittin,
and dermaseptins (16, 28, 31, 36, 37), which demonstrated the changes
in
-helical content in the presence of different concentrations of
TFE and liposomes. The adoption of an amphipathic
-helical structure
in a polar environment or in the presence of liposomes for such
antimicrobial peptides suggests that they could alter the target cell
membrane (23, 38, 39). It is interesting to note that ponericins W are
the only ones that present a hemolytic action, as does melittin, which
acts via the formation of transmembrane pores (16, 40, 41). On the
other hand, ponericins G and L display important similarities with
cecropins and dermaseptins, respectively, which destroy the cell
membrane via a "carpet-like" mechanism (37, 42). Additional NMR
studies and extensive studies of the interactions of the peptides with
liposomes (37) would highlight the structural features and the mode of
action of the ponericins.
The high concentrations of each of the characterized peptides in the
whole venom of P. goeldii (0.33-22.7 mM)
suggest that they could play an important antibacterial role in
vivo. The main function of venom for a predatory ant species is
offensive for prey immobilization and capture. Its defensive role is
generally through predator-prey interactions or competition. But the
antibacterial activities of the peptides isolated from P. goeldii venom highlight another possible aspect of the defensive
function of the venoms. Indeed, the "microbial cleaning" of prey
before their introduction into the colony and their consumption by the
brood ensures colony survival. Thus, the present study suggests that in
predatory ant species, venom may serve to protect against internal
pathogens arising from alimentation, as in the case of peptides in some spider and snake venoms (17, 18).
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ACKNOWLEDGEMENTS |
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We thank the Association pour la Recherche contre le Cancer for financial support toward the purchase of the matrix-assisted laser ionization/desorption time-of-flight mass spectrometer, Dr. P. Bulet (Institute de Biologie Moléculaire et Cellulaire, Strasbourg, France) for expert advice, Dr. M. Guyot (Museum National d'Histoire Naturelle, Paris, France) for assistance, J. Giovannoni (Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, Paris, France) for preliminary experiments, V. Labas (Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris) for sequence analysis, the Laboratoire Environnement de Petit Saut (Electricité de France-Centre National d'Equipement Hydrualique) of French Guiana for logistical help during field missions, Dr. J. H. C. Delabie (Centro de Pesquisas do Cacau, and Comissao Executiva do Plano da Lavoura Cacaueira, Itabuna, Bahia, Brazil) for ant identification, and Andrea Dejean for English correction of the manuscript.
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FOOTNOTES |
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* 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.
The amino acid sequences reported in this paper have been submitted to the Swiss Protein Database under Swiss-Prot accession numbers P82414 (Ponericin G1), P82415 (Ponericin G2), P82416 (Ponericin G3), P82417 (Ponericin G4), P82418 (Ponericin G5), P82419 (Ponericin G6), P82420 (Ponericin G7), P82421 (Ponericin L1), P82422 (Ponericin L2), P82423 (Ponericin W1), P82424 (Ponericin W2), P82425 (Ponericin W3), P82426 (Ponericin W4), P82427 (Ponericin W5), and P82428 (Ponericin W6).
¶ To whom correspondence should be addressed. Tel.: 33-0-1-40-79-47-69; Fax: 33-0-1-40-79-47-57; E-mail: virginie.redeker@espci.fr.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M100216200
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; MIC, minimal inhibitory concentration; TFE, trifluoroethanol.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Fujiwara, S.,
Imai, J.,
Fujiwara, M.,
Yaeshima, T.,
Kawashima, T.,
and Kobayashi, K.
(1990)
J. Biol. Chem.
265,
11333-11337 |
2. | Fernandes, A., Lopes, C. A. M., Sforcin, J. M., and Funari, S. R. C. (1996) J. Venom. Anim. Toxins 3, 287-294 |
3. | Rosengaus, R. B., Guldin, M. R., and Traniello, J. F. A. (1998) J. Chem. Ecol. 24, 1697-1706 |
4. | Hölldobler, B., and Engel-Siegel, H. (1984) Psyche 91, 201-224 |
5. | Maschwitz, U., Koob, K., and Schildknecht, H. (1970) J. Insect Physiol. 16, 387-404 |
6. | Veal, D. A., Trimble, J. E., and Beattie, A. J. (1992) J. Appl. Bacteriol. 72, 188-194[Medline] [Order article via Infotrieve] |
7. | Mackintosh, J. A., Flood, J. A., Veal, D. A., and Beattie, A. J. (1999) Austr. J. Entomol. 38, 124-126[CrossRef] |
8. | Brown, C. A., Watkins, J. F., and Eldridge, D. W. (1979) J. Kansas Entomol. Soc. 52, 119-122 |
9. | Hölldobler, B., and Wilson, E. O. (1990) The Ants , Springer-Verlag, Berlin |
10. | Orivel, J., Souchal, A., Cerdan, P., and Dejean, A. (2000) Sociobiology 35, 131-140 |
11. | Blum, M. S., and Hermann, H. R. (1978) in Arthropod Venoms (Bettini, S., ed) , pp. 801-869, Springer-Verlag, Berlin |
12. | Schmidt, J. O. (1986) in Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioural Aspects (Piek, T., ed) , pp. 425-508, Academic Press, London |
13. | Jouvenaz, D. P., Blum, M. S., and MacConnell, J. G. (1972) Antimicrob. Agents Chemother. 2, 291-293[Medline] [Order article via Infotrieve] |
14. | Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835[CrossRef][Medline] [Order article via Infotrieve] |
15. | Krishnakumari, V., and Nagaraj, R. (1997) J. Peptide Res. 50, 88-93[Medline] [Order article via Infotrieve] |
16. | Juvvadi, P., Vunnam, S., and Merrifield, R. B. (1996) J. Am. Chem. Soc. 118, 8989-8997[CrossRef] |
17. | Blaylock, R. S. M. (2000) Toxicon 38, 1529-1534[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Yan, L.,
and Adams, M. E.
(1998)
J. Biol. Chem.
273,
2059-2066 |
19. |
Seon, A. A.,
Pierre, T. N.,
Redeker, V.,
Lacombe, C.,
Delfour, A.,
Nicolas, P.,
and Amiche, M.
(2000)
J. Biol. Chem.
275,
5934-5940 |
20. |
Casteels, P.,
Ampe, C.,
Jacobs, F.,
and Tempst, P.
(1993)
J. Biol. Chem.
268,
7044-7054 |
21. | Finney, D. J. (1962) Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve , Cambridge University Press, Cambridge |
22. | Manavalan, P., and Johnson, W. C. (1987) Anal. Biochem. 167, 76-85[Medline] [Order article via Infotrieve] |
23. | Boman, H. G., and Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Lowenberger, C.,
Charlet, M.,
Vizioli, J.,
Kamal, S.,
Richman, A.,
Christensen, B. M.,
and Bulet, P.
(1999)
J. Biol. Chem.
274,
20092-20097 |
25. | Sun, D., Eccleston, E. D., and Fallon, A. M. (1998) Biochem. Biophys. Res. Commun. 249, 410-415[CrossRef][Medline] [Order article via Infotrieve] |
26. | Andreu, D., and Rivas, L. (1998) Biopolymers 47, 415-433[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ekengren, S., and Hultmark, D. (1999) Insect. Biochem. Mol. Biol. 29, 965-972[CrossRef][Medline] [Order article via Infotrieve] |
28. | Park, J. M., Jung, J. E., and Lee, B. L. (1994) Biochem. Biophys. Res. Commun. 205, 948-954[CrossRef][Medline] [Order article via Infotrieve] |
29. | Simmaco, M., Mignogna, G., and Barra, D. (1998) Biopolymers 47, 435-450[CrossRef][Medline] [Order article via Infotrieve] |
30. | Banks, B. E. C., and Shipolini, R. A. (1986) in Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioural Aspects (Piek, T., ed) , pp. 329-416, Academic Press, London |
31. | Mor, A., and Nicolas, P. (1994) Eur. J. Biochem. 2, 145-154 |
32. | Amiche, M., Seon, A. A., Pierre, T. N., and Nicolas, P. (1999) FEBS Lett. 456, 352-356[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Ehret-Sabatier, L.,
Loew, D.,
Goyffon, M.,
Fehlbaum, P.,
Hoffman, J. A.,
van Dorsselaer, A.,
and Bulet, P.
(1996)
J. Biol. Chem.
271,
29537-29544 |
34. | Giacometti, A., Cirioni, O., Barchiesi, F., Del Prete, M. S., and Scalise, G. (1999) Peptides 20, 1265-1273[CrossRef][Medline] [Order article via Infotrieve] |
35. | Deleage, G., Blanchet, C., and Geourjon, C. (1997) Biochimie (Paris) 79, 681-686[CrossRef][Medline] [Order article via Infotrieve] |
36. | Houston, M. E., Kondejewski, L. H., Gough, M., Fidai, S., Hodges, R. S., and Hancock, R. E. W. (1998) J. Peptide Res. 52, 81-88[Medline] [Order article via Infotrieve] |
37. |
Wang, W.,
Smith, D. K.,
Moulding, K.,
and Chen, H. M.
(1998)
J. Biol. Chem.
273,
27438-27448 |
38. | Matsuzaki, K. (1999) Biochim. Biophys. Acta 1462, 1-10[Medline] [Order article via Infotrieve] |
39. | Silvestro, L., Gupta, K., Weiser, J. N., and Axelsen, P. H. (1997) Biochemistry 36, 11452-11460[CrossRef][Medline] [Order article via Infotrieve] |
40. | Takei, J., Remenyi, A., Clarke, A. R., and Dempsey, C. E. (1998) Biochemistry 37, 5699-5708[CrossRef][Medline] [Order article via Infotrieve] |
41. | Takei, J., Remenyi, A., and Dempsey, C. E. (1999) FEBS Lett. 442, 11-14[CrossRef][Medline] [Order article via Infotrieve] |
42. | Shai, Y. (1999) Biochim. Biophys. Acta 1462, 55-70[Medline] [Order article via Infotrieve] |