Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4

Peptides 26 (2005) 1113–1119

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4 Karin Thevissen a, ∗ , Isabelle E.J.A. Franc¸ois a , Lolke Sijtsma b , Aart van Amerongen b , Wim M.M. Schaaper c , Rob Meloen c , Truus Posthuma-Trumpie c , Willem F. Broekaert a, 1 , Bruno P.A. Cammue a a

b

Centre for Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium Agrotechnology and Food Innovations (A&F), Wageningen University and Research Center, P.O. Box 17, 6700 AA Wageningen, The Netherlands c Pepscan Systems B.V., Edelhertweg 15, P.O. Box 2098, 8203 AB Lelystad, The Netherlands Received 17 November 2004; received in revised form 13 January 2005; accepted 14 January 2005 Available online 16 February 2005

Abstract Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-d forms. We show that the all-d variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 ␮M. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections. © 2005 Elsevier Inc. All rights reserved. Keywords: Antifungal; Ib-AMP; Ionic strength; Toxicity; Derivative

1. Introduction Plants have been shown to produce a wide range of antimicrobial proteins to defend themselves against phytopathogenic microorganisms. These proteins play a key role in plant defense, both as part of preexisting, developmentally regulated defense barriers and as components of the defense responses induced upon infection (reviewed in [2,17]). Four closely related small peptides (20 amino acid residues) were isolated from seeds of Impatiens balsamina and were shown to be inhibitory to the growth of a range of Gram-positive bacteria and fungi (including the pathogens Candida albicans and Aspergillus flavus) [9,13]. These peptides are ∗

Corresponding author. Tel.: +32 16 32 96 88; fax: +32 16 32 19 66. E-mail address: [email protected] (K. Thevissen). 1 Current address: CropDesign NV, Technologiepark 3, B-9502 Gent, Belgium. 0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.01.008

noncytotoxic to cultured human, insect and plant cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3 and Ib-AMP4, are highly basic (containing 5–6 arginine residues) and contain four cysteine residues which form two intramolecular disulfide bonds. The four mature Ib-AMPs are generated by processing of a single polypeptide precursor [13] in a similar manner as apidaecins, antimicrobial peptides from the honey bee [4,5]. Searches of protein databases have failed to identify any proteins with significant homology to Ib-AMPs. Structural studies of Ib-AMP1 reveal that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. From residue 6–20, the backbone is well defined, forming a distinctive loop structure [11]. Unlike many other small antimicrobial peptides, Ib-AMP1 does not form an amphipatic helix and is unlikely to function by nonspecific pore-formation and membrane disruption. Consistently, Ib-AMP1 induces phospholipid disruption of negatively charged liposomes

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composed of phosphatidylcholine-phosphatidylserine (4:1) to a much lesser extent as cecropin A(1-8)-magainin 2(1-12), an ␣-helical amphipatic antimicrobial peptide [9]. In addition, confocal microscopy showed that Ib-AMP1 binds on the fungal cell surface or penetrates into fungal cell membranes [9]. Whether Ib-AMP1 is subsequently internalized in the fungal cell and interacts with intracellular targets is currently not clear. In the present study, we have produced a synthetic variant of Ib-AMP1, which was oxidized to the bicyclic native conformation, and compared its antifungal activity to that of the native form. In addition, four variants of the Ib-AMP1 and IbAMP4 peptides, namely two linear 20-mer peptides in which the cysteines have been replaced by ␣-aminobutyric acid and two linear 20-mer peptides consisting of d-amino acids with d-␣-aminobutyric acid substitutions for cysteine, were produced. Finally, involvement of arginine (R) and tryptophan (W) in the antifungal activity of Ib-AMP4 was assessed via the production of a set of R- and W-replacement Ib-AMP4 variants. Antifungal activity of all the Ib-AMP variants and native peptides and their hemolytic and toxic activities on erythrocytes and myeloma cells, respectively, was studied.

2. Materials and methods 2.1. Materials The antimicrobial peptides Ib-AMP1 and Ib-AMP4 were isolated from seeds of I. balsamina as described previously [13]. 2.2. Peptide synthesis and peptide cyclization Synthesis of linear and all-d Ib-AMP variants and peptide cyclization were carried out as described previously [12]. We used a Hamilton Microlab 2200 (Reno, NV, USA) for synthesis of peptides at 30 ␮mol scale. The Microlab 2200 was programmed to deliver washing solvents and reagents to 4 ml columns with filter, containing Rink-amide resin for peptide synthesis. The synthesis was based on Fmoc/HBTU chemistry [7] using double coupling steps of 40 min. Peptides were deprotected and cleaved in 2 h using 1.5 ml of a mixture of TFA/phenol/TA/water/EDT (40/3/2/2/1, v/w/v/v/v) and then precipitated twice by adding hexane/diethylether (1/1; v/v). The precipitate was dried and lyophilized from water/acetonitrile (1/1; v/v). Ib-AMP1 was synthesized at 0.25 mmol scale on a ABI 430A synthesizer using the same chemistry as above. The purified linear product was oxidized by air oxidation at 0.5 mg/ml in 1% (w/v) ammoniumbicarbonate/water in 2 days as described earlier [12]. The bicyclic product was purified using preparative HPLC. For analytical HPLC we used two Waters pumps model 510, a Waters gradient controller model 680, a Waters WISP 712 autoinjector, and a Waters 991 photodiode array detector. Products were analyzed in a linear gradient from water

with 0.1% (v/v) TFA to 60% (v/v) acetonitrile/water with ˚ 0.1% (v/v) TFA in 60 min on a Waters Delta Pak C18-100 A (3.9 mm × 150 mm, 5 ␮m) column at 1 ml/min. Preparative HPLC was carried out using a Waters Prep 4000 liquid chromatograph, equipped with a Waters RCM module with two PrepPak cartridges plus guard cartridge (25 mm × 210 mm) ˚ (15 ␮m) material. Peptides filled with Delta-Pak C18-100 A were detected at 230 nm using a Waters 486 spectrophotometer with a preparative cell. A set of linear Ib-AMP4 peptides in which at each position, the amino acids had been replaced systematically by Arginine (R) or Tryptophan (W) were synthesized and purified as described above. Peptide stocks were prepared in isotonic buffer (10 mM Tris/HCl, 150 mM NaCl, pH 7.4) for subsequent testing of their antifungal activity and hemolytic and toxic properties. 2.3. Antifungal activity assay Antifungal activity of protein samples against the fungi Botrytis cinerea JHCC 8973, Fusarium culmorum IMI 180420 and Neurospora crassa FGSC 2489 was assayed by microspectrophotometry of liquid cultures grown in microtiter plates as described previously [3,14]. Growth medium used was 1/2 (half strength) PDB (12 g/l Potato Dextrose Broth, Difco) supplemented with 50 mM HEPESNaOH (pH 7.0). Activity of protein samples against the yeasts Saccharomyces cerevisiae BY4741 (Invitrogen, Carlsbad, CA) and Pichia pastoris GS115 (Invitrogen) was assayed by microspectrophotometry as described previously [15]. Growth medium used was full strength PDB (24 g/l Potato Dextrose Broth, Difco) supplemented with 50 mM HEPES-NaOH (pH 7.0). To increase ionic strength of the growth medium, 5 mM CaCl2 was added. Antifungal activity of the R- and W-replacement peptides was tested against the fungus F. culmorum IMI 180420 in 1/2 PDB, 50 mM HEPES-NaOH (pH 7.0) and in SMF (Synthetic Medium Fungi, [14]), pH 7.0, with different ionic strength. Media used were: (1) SMF+, SMF supplemented with 1 mM CaCl2 , 50 mM KCl; (2) SMF3+, SMF supplemented with 3 mM CaCl2 , 3 mM MgCl2 and 100 mM KCl; (3) SMF supplemented with 10 mM CaCl2 , 3 mM MgCl2 and 100 mM KCl. IC50 values of the peptides (i.e. the peptide concentration required to inhibit 50% of fungal growth) were determined from duplo experiments after 72 h of incubation. 2.4. Hemolytic activity of peptides Hemolytic activity of the peptides was tested on rabbit erythrocytes. Fifty microliters aliquots of the peptides (up to 200 ␮M, generally 100 ␮M) in isotonic buffer A (10 mM Tris/HCl, 150 mM NaCl, pH 7.4) were incubated with 150 ␮l of a pre-washed rabbit erythrocyte solution in buffer A (108 cells/ml) for 24 h at 25 ◦ C. After centrifugation (5 min, 1000 × g), the optical density of the supernatant was

K. Thevissen et al. / Peptides 26 (2005) 1113–1119

measured at 405 nm. A 0% lysis control (without peptide) and a 100% lysis control (fully lysed erythrocytes in water) were used to assess the hemolytic activity of the various peptides.

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Table 1 Amino acid compositions of native and synthetic variants of Ib-AMPs

2.5. Toxicity of peptides Toxicity of the peptides was tested on mouse myeloma cells. Fifty microliters aliquots of the peptides (up to 200 ␮M, generally 100 ␮M) in buffer A (10 mM Tris/HCl, 150 mM NaCl, pH 7.4) were incubated with 150 ␮l of mouse myeloma cells in buffer A (5 × 105 cells/ml) for 24 h in a CO2 incubator at 37 ◦ C. The percentage of living cells as compared to the control (without peptide) was scored colorimetrically after administration of Trypan Blue (0.2% in PBS).

3. Results 3.1. Synthesis of Ib-AMP1 The synthesis of Ib-AMP1 was performed at 0.25 mmol scale by solid phase synthesis on an ABI 430A synthesizer. The resin that was used generated a C-terminal amide peptide after cleavage. The crude linear peptide was purified using preparative HPLC and the purified product was cyclisized at smaller scale by air oxidation at pH 8. The oxidation process was followed by analytical HPLC and mass spectrometry and proceeded very efficiently via the single disulfide product to the bicyclic product. Other oxidation procedures, like DMSO oxidation at pH 5–6, followed the same reaction path, showing the preferred formation of the two disulfide bridges (results not shown). Synthetic Ib-AMP1 was found to be as active on the three test organisms F. culmorum, S. cerevisiae and P. pastoris as native Ib-AMP1 in a medium with low or high ionic strength (results not shown). Moreover, synthetic Ib-AMP1 caused typical branching patterns of F. culmorum as in case of native Ib-AMP1 (see Section 3.3). Overall, antifungal activity of synthetic Ib-AMP1 was found to be similar to native Ib-AMP1 on both, fungi and yeast strains. 3.2. Structural analysis of native and synthetic variants of Ib-AMP1 and Ib-AMP4 Structural analysis of Ib-AMP1 showed that the four cysteine residues form two intramolecular disulfide bonds, namely between Cys-6 and Cys-16 and between Cys-7 and Cys-20 [11]. In order to test whether the loop structure is important for the antifungal activity of Ib-AMPs, linear variants of Ib-AMP1 and Ib-AMP4 (MCE01 and MCE02, respectively) were synthesized in which cysteines were replaced by the isosteric ␣-aminobutyric acid (Table 1). NMR data showed that the non-disulfide-linked peptide adopts a random coil conformation (Dr. Fant, NMR and Structure Analysis Unit, Department of Organic Chemistry, Faculty of Sciences,


Ghent University, Belgium, personal communications), confirming that the loop conformation adopted by the peptide is due to the disulfide restraints. In addition, linear all-d amino acid peptide analogues of Ib-AMP1 and Ib-AMP4 with d␣-aminobutyric acid substitutions (MCD26 and MCD30, respectively) were synthesized in order to elucidate whether Ib-AMPs are interacting in a stereospecific way with fungal target components. 3.3. Antifungal activity of native and linear 20-mer variants of Ib-AMP1 and Ib-AMP4 The antifungal activity of native and synthetic linear 20mer Ib-AMP-variants was assessed on three fungal and two yeast strains (Table 2). In buffered low ionic strength medium, IC50 values of native Ib-AMPs on fungi were <3 ␮M. Yeast strains were a factors 10–20 more resistant to native IbAMPs than fungal strains. The linear all-d Ib-AMP-variants, MCD26 and MCD30, were as active as native Ib-AMPs on fungi, whereas these variants were a factors 2–8 more active on yeast strains as compared to native Ib-AMPs. Overall, all-l amino acid peptides were a factors 2–6 less active as compared to all-d amino acid peptides, and Ib-AMP4 showed the highest activity on the majority of the fungal and yeast strains. It was shown previously that the antifungal activity of the native Ib-AMPs is dependent on the ionic strength of the growth medium [13]. This is in accordance with our observations that antifungal activity of native and synthetic Ib-AMP variants was severely reduced in buffered media with high ionic strength (factors 5–50) (Table 2). Whether this decrease in antifungal activity of the peptides is due to the presence of specific cations or the general increase in ionic strength is currently not clear. Only native Ib-AMP4 maintained significant inhibitory activity on N. crassa, and the linear all-d peptides MCD26 and MCD30 remained active on P. pastoris in this medium. In contrast to the synthetic Ib-AMP-variants, native IbAMPs caused swelling and branching of hyphae of particular fungi, such as N. crassa and F. culmorum, irrespective of

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Table 2 Antifungal activity of native and synthetic variants of Ib-AMPs against different fungi in media with different ionic strengths Fungus

IC50 a (␮M) Ib-AMP1

N. crassa B. cinerea F. culmorum S. cerevisiae P. pastoris

Ib-AMP4

MCD26

MCD30

MCE01

MCE02

−b

+ISc

−

+IS

−

+IS

−

+IS

−

+IS

−

+IS

0.5 1.5 1.4 15 16

8 50 >50 50 >50

1.2 3 1.0 13 5

4 20 15 >50 40

0.8 0.5 1.4 2 2

16 25 50 >25 3

1.5 1 0.5 7.5 1

16 25 10 >25 3

3 1.5 5 20 6

22 25 >50 >25 15

3 2 5 20 4

20 25 35 >25 15

Data are means of duplicate measurements. S.E. were typically below 6.5%. a IC value (i.e. the concentration of the antifungal protein that is required to inhibit 50% of the growth) is expressed as means of duplicate experiments. 50 b No increase in ionic strength of the growth medium, which was 1/2 PDB for fungal strains and PDB, pH 7.0 for yeast strains. c Addition of 5 mM CaCl to increase ionic strength of the growth medium. 2

the ionic strength (or the presence of specific cations) of the medium (results not shown). 3.4. Antifungal activity of R- and W-replacement Ib-AMP4 variants Via mutational analysis of the plant defensin Rs-AFP2, a small (51 amino acid residues) antifungal peptide from seeds of Raphanus sativus [14], we showed previously that the RsAFP2 antifungal activity can be substantially increased by 200–250% via replacement of glycine at position 9 or valine at position 39 by arginine (R) thereby increasing net positive charge (in the physiological pH range) [6]. Replacement of serine at position 21, leucine at position 28, alanine at position 42 or isoleucine at position 46 with arginine on the other hand, decreased the antifungal activity of Rs-AFP2. Whether this is due to the unfavorable presence of an extra charge or to the replacement of an amino acid essential for the antifungal activity is currently not clear. In addition, we showed previously that replacement of alanine at position 31 by tryptophan (W) results in an Rs-AFP2 form with nine-fold decrease in antifungal activity. Substitution of the alanine by a bulky tryptophan residue most probably results in a conformational distortion, which might explain the drastic reduction of the antifungal activity of this variant [6]. To determine whether introduction of R or W affects the antifungal activity of Ib-AMPs, two series of Ib-AMP4 peptide derivatives were synthesized in which each amino acid was substituted, one at a time, by R or W. We chose Ib-AMP4 since this peptide is the most active against the majority of fungal and yeast strains tested (Table 2). In Tables 3 and 4, the antifungal activity of these R- and W-derivatives of Ib-AMP4 is compared to the activity of native Ib-AMP4 and linear Ib-AMP4 (MCE02). Native Ib-AMP4 was found to be most active against F. culmorum as compared to each of the R-substituted Ib-AMP4 peptide derivatives (Table 3). Introduction of R residues in the Ib-AMP4 sequence did not influence the antifungal activity of the peptide derivatives by more than two-fold as compared to Ib-AMP4. Four of the R-replacement peptides, namely MCC03, MCC04, MCC08 and MCC14 showed more than

a two-fold increased salt-dependency of their activity as compared to Ib-AMP4. None of the R-replacement peptides, however, showed a decreased salt-dependency of their activity by more than two-fold as compared to Ib-AMP4. Similarly, the W-substituted Ib-AMP4 peptide variants did not show improved antifungal activity nor saltdependency by more than two-fold as compared to native Ib-AMP4 (Table 4). One W-substituted Ib-AMP4 variant, namely MCC18, showed more than two-fold increased saltdependency of its antifungal activity as compared to native Ib-AMP4. Ib-AMP1, Ib-AMP4 and five Ib-AMP4 variants of the Wscan were selected, based on differential salt-dependency of their antifungal activity, and retested in media with increasing ionic strength (Table 5). Peptide MCC17 retained activity even in the buffered SMF10+ medium (SMF with 10-fold increase in ionic strength). Native Ib-AMP4 was still active in the buffered SMF3+ medium, whereas native Ib-AMP1 was only active in the medium with the lowest ionic strength, i.e. SMF+.

Table 3 Antifungal activity of Ib-AMP4, MCE02 and Ib-AMP4 variants of the Rreplacement scan against F. culmorum in 1/2 PDB and SMF+, pH 7.0


K. Thevissen et al. / Peptides 26 (2005) 1113–1119 Table 4 Antifungal activity of Ib-AMP4, MCE02 and Ib-AMP4 variants of the Wreplacement scan against F. culmorum in 1/2 PDB and SMF+, pH 7.0

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wards mouse myeloma cells (Table 6). Both native proteins, Ib-AMP1 and Ib-AMP4, did not show any hemolytic or toxic activity up to a concentration of 100 ␮M. In contrast, increased hemolysis and toxicity could be detected with Ib-AMP4 variants, with MCC17 being the most active.

4. Discussion



3.5. Hemo(lytic) and toxic activity of Ib-AMP1, Ib-AMP4 and selected W-scan Ib-AMP4-derivatives The toxicity of several native Ib-AMPs was previously investigated on human skin fibroblasts up to a concentration of 100 ␮M. None of these peptides did perturb membrane integrity and thus, Ib-AMPs were considered non-toxic [13]. In this study, we assessed the hemolytic activity of Ib-AMP1, Ib-AMP4 and selected W-scan Ib-AMP4-derivatives (see Section 3.4) on rabbit erythrocytes and their toxic activity toTable 6 Hemolytic and toxic activity of Ib-AMP4 variants of the W-replacement scan


This study describes the analysis of the antifungal activity of the antimicrobial peptides Ib-AMP1 and Ib-AMP4, isolated from I. balsamina seeds. It was shown previously that the antifungal activity of these peptides can be drastically reduced in the presence of cations [13]. The aim of the present study was to synthetically produce native Ib-AMPs and to produce Ib-AMP variants with higher antifungal activity or decreased salt-dependency as compared to native Ib-AMPs. We first demonstrated that, although some improvements in yield can still be made, the synthesis strategy for Ib-AMP1 makes an efficient synthesis of Ib-AMPs possible, which retain their antifungal activity against a variety of fungal and yeast strains. In addition, structure analysis of synthetic Ib-AMP1 proved that both disulfide bridges were formed correctly. To obtain Ib-AMP variants with improved antifungal activity or decreased salt-dependency, we produced in a first instance, linear all-l and all-d amino acid variants of Ib-AMP1 and Ib-AMP4. We show that all-d linear peptide variants of the Ib-AMPs are as active on yeast and fungal strains as native peptides, indicating that the Ib-AMPs are probably not interacting with proteinaceous receptors on the fungal plasma membrane in a stereospecific manner. In addition, we demonstrate that the antifungal activity of native and the tested synthetic Ib-AMP variants is severely reduced in media with high ionic strength. This is in accordance with previous results on the salt-dependency of native Ib-AMPs [13]. Such antagonizing effect of divalent cations on the activity of antifungal components has been reported previously for various membrane-interacting antifungal peptides, such as defensins from plants, human and insects (reviewed in [17]). Cations, especially divalent cations, stabilize membrane phospholipid structures and hence, they can prevent the induced membrane permeabilization. In a second instance, we produced an R- and Wreplacement scan of Ib-AMP4, which is the most active and less salt-sensitive of the Ib-AMPs. This approach was inspired by a mutational analysis of the plant defensin Rs-AFP2, isolated from seed of Raphanus sativus [6]. In this analysis, it was shown that the antifungal activity of Rs-AFP2 can both be increased and decreased via replacement of certain amino acid residues by arginine, thereby increasing the net positive charge. However, the activity of none of the R-replacement peptides was affected by more than two-fold as compared to native Ib-AMP4. Notably, two of the R-replacement peptides, namely MCC03 (W at position 2 replaced by R) and MCC14 (W at position 19 replaced

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by R), both with replacement of W by R, showed more than a three-fold increased salt-dependency of their activity as compared to Ib-AMP4. Interestingly, replacement of the other W at position 9 by R also increased salt-dependency by two-fold. Thus, it seems that replacement of either of the tryptophan residues in Ib-AMP4 by arginine leads to increased salt-dependency of the antifungal activity. None of the R-replacement peptides, however, showed a decreased salt-dependency of their activity by more than two-fold as compared to Ib-AMP4. Furthermore, we have previously shown that replacement of certain amino acid residues by the bulky tryptophane residue in Rs-AFP2, can decrease its antifungal activity. Remarkably, this loss in antifungal potency of Rs-AFP2 was less noticeable in media with low ionic strength [6]. In accordance with this study, one W-substituted Ib-AMP4 variant, namely MCC18 (R at position 13 replaced by W), showed more than two-fold increased salt-dependency of its antifungal activity as compared to native Ib-AMP4. However, none of the W-substituted Ib-AMP4 peptide variants showed decreased antifungal activity by more than two-fold as compared to native Ib-AMP4. Five W-derivatives of Ib-AMP4 were selected and their antifungal activity was tested in media with very high ionic strength. Interestingly, MCC17, an Ib-AMP4 derivative having a W residue for glycine at position 3 retained its antifungal activity even in the buffered SMF10+ medium (synthetic medium with 10-fold increased ionic strength). However, MCC17 showed the highest hemolytic and toxic activity as compared to the native peptides, indicating that medicinal use of this peptide is excluded. In conclusion, the R- and W-scans of Ib-AMP4 show that improvement of inhibitory activity as compared to the native protein is not possible with these substitutions in media with low ionic strength. However, improvements in salt-dependency in relation to Ib-AMP4 can be obtained in media with high ionic strength, as for MCC17. Unfortunately, this peptide has high toxic and hemolytic activity. Thus, with respect to both antifungal properties and absence of toxic side-effects, of all tested natural and synthetic Ib-AMP forms, Ib-AMP4 seems to be the best candidate for antimycotic applications to combat fungal diseases. In contrast to the synthetic Ib-AMP-variants, native Ib-AMPs caused swelling and branching of hyphae of particular fungi, such as N. crassa and F. culmorum, irrespective of the ionic strength of the medium. Similar phenotypic characteristics, i.e. extensive branching of fungal hyphae, have previously been observed in case of incubation of F. culmorum with Rs-AFP2 [14]. Rs-AFP2 has been shown to permeabilize fungal membranes after an initial interaction with specific binding sites on the membrane, which were recently characterized as glucosylceramides [15,16]. Yeast mutants affected in the biosynthesis of glucosylceramides are resistant to Rs-AFP2 as compared to the corresponding wild-type yeast strain [16]. Glucosylceramides are a class of complex

sphingolipids, which are an important class of membrane components that ensure structural and functional characteristics of the membrane [1]. Increasingly, sphingolipids and their metabolites, such as ceramide and phytosphingosine, are emerging as bioactive signaling molecules that affect events ranging from apoptosis to the regulation of the cell cycle and stress responses [8,10]. It was previously shown that Ib-AMP1 binds to fungal cell surface or penetrates into fungal cell membranes [9]. However, it seems that Ib-AMPs are not interacting with the Rs-AFP2 binding sites as yeast mutants which are lacking glucosylceramides are as susceptible to Ib-AMPs as the corresponding wild-type yeast strains (results not shown). Whether other classes of sphingolipids than glucosylceramides are implicated in the antifungal activity of Ib-AMPs needs to be investigated further.

Acknowledgements This work was supported by a grant from EU (AIR2CT94-1356) and FWO-Vlaanderen (G.0288.04). I.E.J.A.F. (CMPG) acknowledges the receipt of a postdoctoral grant from the IWT-Vlaanderen (Grant OZM/030508). L.B.J.M. Berendsen (A&F) is acknowledged for her contribution to part of the experimental work.

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