Ponericins, New Antibacterial and Insecticidal Peptides from the Venom of the Ant Pachycondyla goeldii*

Jérôme OrivelDagger , Virginie Redeker§, Jean-Pierre Le Caer§, François Krier||, Anne-Marie Revol-Junelles||, Arlette Longeon**, Alain ChaffotteDagger Dagger , Alain Dejean§§, and Jean Rossier§

From the Dagger  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, Dagger Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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.

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. ---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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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."


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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.

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.

                              
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Table I
Antimicrobial and hemolytic action spectra of 10 synthetic peptides from P. goeldii venom
For each peptide, the tested concentration was 0.4-0.5 mM, and the values correspond to the diameters (in mm) of the zone of inhibition.

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).

                              
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Table II
Antibacterial action of ponericins G1, G6, W1, W3-desK, and L2
Cecropin B, melittin, and dermaseptin were used as reference peptides in the plate growth inhibition assay.

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.

                              
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Table III
Insecticidal action (in µg venom/gram injected individual) of the 10 synthetic peptides on the cricket, Acheta domesticus and ant workers of P. goeldii

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.

                              
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Table IV
Quantity and concentration of the 10 synthesized peptides present in one venom reservoir of P. goeldii
The amount of peptide present in one venom reservoir was determined by comparing the area of the HPLC fractions corresponding to each natural peptide to the peak area of a known amount of synthetic peptide. The concentration of each peptide was calculated with a mean volume of the venom reservoir of 0.172 µl, calculated by assimilating the reservoir to an ellipsoid.

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.


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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

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.


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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 alpha -helix or two alpha -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 alpha -helical structure. CD spectra performed in the far-ultraviolet confirmed the adoption of an alpha -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 alpha -helical content in the presence of different concentrations of TFE and liposomes. The adoption of an amphipathic alpha -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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

The abbreviations used are: HPLC, high pressure liquid chromatography; MIC, minimal inhibitory concentration; TFE, trifluoroethanol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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