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Originally published In Press as doi:10.1074/jbc.M312260200 on January 6, 2004

J. Biol. Chem., Vol. 279, Issue 13, 12277-12285, March 26, 2004
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A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid*

Dorit Avrahami and Yechiel Shai{ddagger}

From the Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, November 10, 2003 , and in revised form, January 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing D- and L-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K4X7W, where X designates Gly, Ala, Val, or Leu (designated D-X peptides). The data revealed that PA-D-G and PA-D-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-D-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both D-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-D-L and PA-D-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Together with the growing number of individuals with impaired host defenses, invasive mycoses have emerged as major causes of morbidity and mortality in the last decade (1–5). Although the spectrum of fungal pathogens has changed, the vast majority of invasive fungal infections are still due to Aspergillus and Candida species (4–6). Because of their eukaryotic nature fungal cells have only a restricted set of unique targets. This makes it difficult to selectively target fungal cells. Two major families have been used for more than two decades to combat fungi; these include azoles, which inhibit sterol formation, and polyenes, which bind to mature membrane sterols. However, the development of fluconazole resistance among different pathogenic strains and the high toxicity of amphotericin B (7–9) have prompted the studies of new antifungal agents with new modes of actions.

The investigation of antimicrobial peptides, from a wide range of biological sources, and their synthetic derivatives is a novel inroad to new antifungal agents. Antimicrobial peptides are gene-encoded and are part of the innate immunity to the microbial invasion of microorganisms of all types. The most studied group are short (<40 amino acids), positively charged, linear peptides whose possible mechanisms have been reviewed in detail (10–16). It is believed that most of these peptides are targeted to biological membranes, increase their permeability, and kill the cells. However, other additional mechanisms were proposed for some of them (17). Studies that investigated the mode of action of native antimicrobial peptides that are also endowed with antifungal activity are limited and included, for example, LL-37 and dermaseptines (18–20). These studies showed that they self-associate in solution and reach the membrane as oligomers (18–20).

Previously, we reported on a new approach for increasing the hydrophobicity and self-assembly in a solution of antibacterial peptides that are not active on fungi (21). We showed that peptide oligomerization in solution is crucial for the peptide to endow antifungal activity. In another study we examined the effect of multiple substitutions of different amino acids regarding the structure, binding, and biological function of positively charged diastereomers (include both D- and L-amino acids) of linear lytic peptides (22). For this purpose, a group of diastereomeric peptides with the sequence K4X7W were synthesized, where X designates one of the aliphatic amino acids, Gly, Ala, Val, or Leu. The results revealed that the peptides containing Ala and Gly are not active against microorganisms, concomitant with their inability to bind membranes. This is in agreement with the notion that a threshold of hydrophobicity along with a defined structure is required for antimicrobial activity. Here, we conjugated palmitic acid to this series of diastereomers and investigated their biological function and mode of action. Very interestingly, we found that the most potent antibacterial and antifungal lipopeptides are those derived from the peptides containing Ala and Gly. Studies on the function and structure of the peptides together with their interaction with bacteria, fungi, and model membranes shed light on their mode of action. Furthermore, these studies demonstrated that a lipophilic tail can compensate for the hydrophobicity and amphipathic structure of the peptidic chain previously shown to be a prerequisite for antimicrobial activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—4-Methylbenzhydrylamine (MBHA) Rink amide resin was obtained from Calbiochem-Novabiochem AG (Switzerland). Other reagents used for peptide synthesis include trifluoroacetic acid (Sigma), piperidine (Merck), N,N-diisopropylethylamine (Sigma), N-hydroxybenzotriazole hydrate (Aldrich), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and dimethylformamide (peptide synthesis grade, Bio-lab Ltd.). Egg phosphatidylcholine (PC),1 phosphatidylinositol (PI), and phosphatidylethanolamine (PE) ergosterol were purchased from Lipid Products (South Nutfield, UK). 3-3'-Dipropylthiadicarbocyanine iodide (diS-C3-5) and calcein were purchased from Molecular Probes (Junction City, OR). All other reagents were of analytical grade. Buffers were prepared in double-distilled water. Amphotericin B and trypsin (bovine pancreas) were purchased from Sigma. RPMI 1640 was purchased from Biological Industries (Beit Haemek, Israel). Proteinase K (fungal) was purchased from Beckman Instruments.

Peptide Synthesis, Acylation, and Purification—Peptides were synthesized by a Fmoc solid phase method on Rink amide 4-methylbenzhydrylamine resin using an ABI 433A automatic peptide synthesizer. The lipophilic acid was attached to the N terminus of a resin-bound peptide using standard Fmoc chemistry. Briefly, after removal of the Fmoc from the N terminus of the peptide with a solution of 20% piperidine in dimethylformamide, the fatty acid (7 eq, 1 M in dimethylformamide)) was coupled to the resin under similar conditions used for the coupling of an amino acid. The peptides were cleaved from the resin by 95% trifluoroacetic acid and purified by reverse phase high performance liquid chromatography (HPLC) on a C18 (for non-lipidic peptides) or C4 (for the lipopeptides) Bio-Rad semi-preparative column (250 x 10 mm, 300-Å pore size, 5-µm particle size). The purified peptides were shown to be homogeneous (>99%) by analytical reverse phase HPLC. The elution time of the lipopeptides increased by ~20 min, indicating an increase in hydrophobicity owing to the attachment of the fatty acid. Electrospray mass spectroscopy was used to confirm their molecular weight, and amino acid analysis was used to confirm the composition of the peptidic moiety.

Antifungal Activity—The antifungal activity of the peptides and their lipophilic acid conjugated analogs was preformed according to the conditions of National Committee for Clinical Laboratory Standards document M27-A. The peptides were examined in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 200 µl as follows. 100 µl of a suspension containing fungi at a concentration of 2 x 103 colony-forming units/ml in culture medium (RPMI 1640, 0.165 M MOPS, pH 7.0, with L-glutamine, without NaHCO3 medium) was added to 100 µl of water containing the peptide in serial 2-fold dilutions. The fungi were incubated for 24 h for opportunistic fungi or 48–72 h for Candida albicans and Cryptococcus neoformans at 35 °C using a Binder KB115 incubator. Growth inhibition was determined by measuring the absorbance at 620 nm in a microplate autoreader El309 (Bio-tek Instruments). Antifungal activity is expressed as the minimal inhibitory concentration (MIC), the concentration at which no growth was observed. The fungi used were Aspergillus fumigatus ATCC 26430, Aspergillus flavus ATCC 9643, Aspergillus niger ATCC 9642, C. albicans ATCC 10231, and C. neoformans ATCC MYA-422.

Antibacterial Activity—The antibacterial activity of the peptides was examined in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 µl as follows. Aliquots (50 µl) of a suspension containing bacteria at a concentration of 106 colony-forming units/ml in culture medium (LB medium) were added to 50 µl of water containing the peptide in serial 2-fold dilutions in water (prepared from a stock solution of 1 mg/ml peptide in water). Inhibition of growth was determined by measuring the absorbance at 492 nm with a Microplate autoreader El309 (Bio-tek Instruments) after an incubation of 18–20 h at 37 °C. Antibacterial activities were expressed as the MIC, the concentration at which no growth was observed after 18–20 h of incubation. The bacteria used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Bacillus subtilis ATCC 6051, and Staphylococcus aureus ATCC 6538P.

Hemolysis of Human Red Blood Cells (hRBC)—Fresh hRBC with EDTA were rinsed 3 times with PBS (35 mM phosphate buffer/0.15 M NaCl, pH 7.3) by centrifugation for 10 min at 800 x g and resuspended in PBS. Lipopeptides dissolved in PBS were then added to 50 µl of a solution of the stock hRBC in PBS to reach a final volume of 100 µl (final erythrocyte concentration, 4% v/v). The resulting suspension was incubated with agitation for 60 min at 37 °C. The samples were then centrifuged at 800 x g for 10 min. The release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for 0 hemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBS and Triton 1%, respectively.

Preparation of Liposomes—Small unilamellar vesicles were prepared by sonication as described earlier (23). Briefly, dry lipids were dissolved in CHCl3:MeOH (2:1 v/v). The solvents were then evaporated under a stream of nitrogen and then lyophilized overnight. The lipids were resuspended in the appropriate buffer (7 mg/ml) with vortexing, and the resulting lipid dispersions were sonicated (10–30 min) in a bath-type sonicator (G1125SP1 sonicator, Laboratory Supplies Company Inc.), until the turbidity had cleared. The vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan). Lipid films were prepared from a mixture of zwitterionic phospholipids and ergosterol (PC/PE/PI/ergosterol, 5:2.5: 2.5:1 w/w), a lipid composition used to mimic the major components of the outer leaflet of C. albicans (24).

Circular Dichroism (CD) Spectroscopy—The CD spectra of the lipopeptides were measured with a Aviv 202 spectropolarimeter. The spectra were scanned with a thermostatic quartz optical cell with a path length of 1 mm. Each spectrum was recorded in an average time of 5 s at steps of 0.2 nm at a wavelength range of 260 to 190 nm. The lipopeptides were scanned at a concentration of 10–100 µM in PBS (35 mM phosphate buffer, 0.15 M NaCl, pH 7.3) and 100 µM in the presence of 1% lysophosphatidylcholine micelles. Fractional helicities (25, 26) were calculated as,

(Eq. 1)
where [{theta}]222 is the experimentally observed mean residue ellipticity at 222 nm, and values for and , corresponding to 0 and 100% helix content at 222 nm, are estimated to be –2,000 and –32,000 deg·cm2/dmol, respectively (25).

In Vivo Transmembrane Potential Depolarization Assay in Bacteria and Yeast—Membrane destabilization, which results in the collapse of transmembrane potential, was detected fluorimetrically by using a fluorescence dye (27) (see details below for bacteria and yeasts). The dye binds the plasma membrane owing to the cell transmembrane potential, resulting in a quenching of the dye fluorescence. Peptide-induced membrane permeation caused a dissipation of the transmembrane potential that was monitored by an increase in fluorescence due to release of the dye. Experiments were performed in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 µl as follows. 50 µl of the cell suspensions and the dye were added to 50 µl of water containing the peptide in serial 2-fold dilutions. Membrane depolarization was monitored by an increase in the fluorescence of diS-C3-5 (excitation wavelength {lambda}ex = 622 nm, emission wavelength {lambda}em = 670 nm).

Transmembrane Potential Depolarization on the yeast C. neoformans—The assay was performed with intact yeast. More specifically, cells were centrifuged at 4000 x g for 5 min at 4 °C in a SS-34 rotor after incubation at 35 °C with agitation for 24 h in RPMI buffer (RPMI 1640, 0.165 M MOPS, pH 7.0, with L-glutamine and without NaHCO3). The cells were resuspended in PBS (without Ca2+ and Mg2+) to an inoculum of 4 x 103 colony-forming units/ml. Cells were incubated with 1 µM diS-C3-5 followed by fluorescence dequenching until a stable base line was achieved (~50 min), indicating the incorporation of the dye onto the yeast membrane.

Transmembrane Potential Depolarization in E. coli—Spheroplasts of the Gram-negative bacteria E. coli D21 were prepared by the osmotic shock procedure (28). Bacteria were grown at 37 °C with agitation to the mid-log phase. Cells from cultures grown (A600 = 0.8) were harvested by centrifugation and washed twice with 10 mM Tris/H2SO4, 25% sucrose, pH 7.5. Cells were resuspended in the washing buffer containing 1 mM EDTA. After 10 min of incubation at 20 °C with rotary mixing, the cells were collected by centrifugation and resuspended immediately in cold (0 °C) water. After 10 min of incubation at 4 °C with rotary mixing, the spheroplasts were collected by centrifugation. The spheroplasts were then resuspended to A600 of 0.05 in a buffer containing 20 mM glucose, 5 mM HEPES, 1 M KCl, pH 7.3. The cells were incubated with 1 µM diS-C3-5 followed by fluorescence dequenching until a stable base line was achieved (~2 h), indicating the incorporation of the dye into the bacteria membrane.

ATR-FTIR Spectroscopy—Spectra were obtained with a Bruker equinox 55 FTIR spectrometer equipped with a deuterated triglyceride sulfate detector and coupled with an ATR device after removing the trifluoroacetate (CF3COO–) counterions, as has been described in detail in other studies (21). The sample was hydrated by exposure of the samples to an excess of deuterium oxide (D2O). The H/D exchange was considered complete due to the complete shift of the amide II band. Any contribution of D2O vapor to the absorbance spectra near the amide I peak region was eliminated by subtraction of the spectra of pure lipids equilibrated with D2O under the same conditions.

ATR-FTIR Data Analysis—To resolve overlapping bands, we processed the spectra using PEAKFITTM (Jandel Scientific, San Rafael, CA) software. Second-derivative spectra were calculated to identify the positions of the component bands in the spectra. These wave numbers were used as initial parameters for curve fitting with gaussian component peaks. Positions, bandwidths, and amplitudes of the peaks were varied until a good agreement between the calculated sum of all components and the experimental spectra was achieved (r2 > 0.999). The relative amounts of different secondary structure elements were estimated by dividing the areas of individual peaks assigned to a particular secondary structure by the whole area of the resulting amide I band.

Analysis of the Polarized ATR-FTIR Spectra—The ATR electric fields of incident light were calculated (29, 30) as follows.

(Eq. 2)

(Eq. 3)

(Eq. 4)
where {theta} is the angle of a light beam to the prism normal at the point of reflection (45°), and n21 = n2/n1 (n1 and n2 are the refractive indices of ZnSe, taken as 2.4, and the membrane sample, taken as 1.5, respectively). Under these conditions, Ex, Ey, and Ez are 1.09, 1.81, and 2.32, respectively. The electric field components together with the dichroic ratio (defined as the ratio between absorption of parallel (to a membrane plane), Ap, and perpendicularly polarized incident light, As) are used to calculate the orientation order parameter, f, by the formula

(Eq. 5)
where {alpha} is the angle between the transition moment of the amide I vibration of an {alpha}-helix and the helix axis. Several values ranging from 27° to 40° were reported in the literature for {alpha} (31). We used the values of 27° (29, 32) and 39° (33) for {alpha}. Lipid order parameters were obtained from the symmetric (~2853 cm–1) and antisymmetric (~2922 cm–1) lipid stretching modes using the same equations, setting {alpha} = 90° (29).

Tryptophan Fluorescence Measurements—To determine the environment of the peptides we measured changes in the intrinsic tryptophan fluorescence in PBS and upon binding to vesicles. A peptide (1 µM) was added to PBS or PBS containing 1 mM PC/PE/PI/ergosterol (5:2.5:2.5:1) small unilamellar vesicles. Emission spectra were measured on a SLM-Aminco Series 2 Spectrofluorimeter with the excitation set at 280 nm using a 4-nm slit and recorded in the range of 300–400 nm (4-nm slit). In these studies small unilamellar vesicles were used to minimize differential light-scattering effects, (34) and the lipid/peptide molar ratio was kept high (1000:1) so spectral contributions of free peptide would be negligible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Peptide Design and Sequences
Four diastereomeric peptides were synthesized corresponding to the sequence KX3KWX2KX2K (X = Gly, Ala, Val, or Leu). They were designed to create a perfect amphipathic {alpha}-helical structure in their L form, as revealed by the Schiffer and Edmundson wheel projection (35). Each diastereomer contained four D-amino acids at positions 3, 4, 8,10, when X = Ala, Val, or Leu, or alternatively at positions 1, 5, 9, 12, when X = Gly. A Trp was introduced in each peptide to serve as an intrinsic fluorescent probe. All four peptides were amidated at their C termini and conjugated at their N termini to palmitic acid (PA), a linear saturated chain of 16 carbons (CH3(CH2)14COOH). Table I shows the sequence of the peptides and their designations and molecular weights.


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  		TABLE I
Peptide sequences and designations

Bold and underlined letters are D-amino acids. The C terminus of all the peptides is amidated.

 
Antibacterial and Antifungal Activity
Antibacterial activity was assayed against three strains of Gram-negative (P. aeruginosa, A. baumannii, and E. coli) and two strains of Gram-positive (S. aureus, B. subtilis) bacteria. Table II presents the MIC of the peptides against the tested bacteria. The data revealed that both D-V and its palmitoylated analog were devoid of antibacterial activity. However, in contrast to D-V, D-L was highly active toward all bacteria tested, but its palmitoylated analog was also devoid of antibacterial activity. Most interestingly, whereas both D-G and D-A were devoid of antibacterial activity, their palmitoylated analogs PA-D-G and PA-D-A were highly active toward all the bacteria tested.


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  		TABLE II
MIC of the peptides against bacteria

For peptides sequences see Table I. ND, not determined.

 
We further tested the antifungal activity of the peptides against representative pathogenic yeasts and fungi. The list includes C. albicans, C. neoformans, A. fumigatus, A. flavus, and A. niger. The results are summarized in Table III. The data revealed that in all cases palmitoylation significantly increased the antifungal activity. Similar to the antibacterial activity, the lipopeptides PA-D-G and PA-D-A were the most active toward all the tested fungi and yeast, whereas the hydrophobic lipopeptides, PA-D-V and PA-D-L, are only partially active.


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  		TABLE III
MIC of the peptides against yeasts and fungi

For peptide sequences see Table I.

 
Hemolytic Activity of the Lipopeptides
The hemolytic activity of the lipopeptides against a highly diluted solution of human erythrocytes (4%) is shown in Fig. 1. The data revealed that PA-D-G and PA-D-A, which were highly active against bacteria, yeasts, and fungi showed low hemolytic activity. In contrast, PA-D-V and PA-D-L, which were practically devoid of antibacterial activity and only partially killed yeasts and fungi, were highly hemolytic.



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  		FIG. 1.
Dose response of the hemolytic activity of the lipopeptides toward hRBC. The assay was performed as described under "Experimental Procedures." {triangleup}, PA-D-G; {square}, PA-D-A; {blacktriangleup}, PA-D-V; {blacksquare}, PA-D-L. The results are the mean of three separate experiments.

 
Oligomerization of the Lipopeptides in Solution Detected by Tryptophan Fluorescence and CD Spectroscopy
We monitored the fluorescence emission spectrum of the intrinsic tryptophan in aqueous solution. We found that the signal of the Trp emission in PA-D-V was fully quenched (Table IV), possibly because of close proximity between the tryptophans, which causes self-quenching. Because tryptophan is located along the peptide chain, we can assume that the peptidic moiety exists as an oligomer in solution, as suggested previously for the non-lipidic form (22). The data also revealed a significant blue shift in the case of PA-D-L, which suggests that the peptide is located in a more hydrophobic environment. This could possibly happen if the peptide interacts with the lipid moiety, either by bending on itself, as suggested previously with the all L-amino acid magainin derivative (21), or alternatively, because of the formation of micelles in which the peptide is bound to the lipidic moiety. In contrast, no blue shift was observed for PA-D-G and PA-D-A, which suggests that these lipopeptides are either monomeric or oligomerize through the lipidic moiety, whereas the Trp environment is exposed to the solution.


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  		TABLE IV
Tryptophan emission maxima (in nm) of the lipopeptides in aqueous solution (PBS) or in the presence of PC/PE/PI/ergosterol (5:2.5:2.5:1 w/w) vesicles

 
To further support our findings we measured the effect of the lipophilic moiety on the structure of the peptides in PBS as measured by CD spectroscopy (Fig. 2). Dose-dependent (from 10 µM to 100 µM) CD spectral profiles of the lipopeptides showed that only PA-D-L adopts a well defined {alpha}-helical structure, which is concentration-independent. A peptide can form a distinct secondary structure in solution either if it oligomerizes such that the hydrophobic surfaces are packed one against another and the hydrophilic surfaces face the solution or, alternatively, if it is inserted into a hydrophobic environment that could be produced by micellization of the palmitoyl groups. KL-containing diastereomeric peptides were shown previously to be monomeric in solution (22). Furthermore, the blue shift in Trp fluorescence (see above in the previous paragraph) revealed that the peptide is located in a hydrophobic environment. These data support the second possibility, suggesting the formation of mixed micelles.



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  		FIG. 2.
CD spectra of the lipopeptides in PBS. Spectra were taken at a lipopeptide concentration of 100 µM. The assay was performed as described under "Experimental Procedures." {triangleup}, PA-D-G; {square}, PA-D-A; {blacktriangleup}, PA-D-V; {blacksquare}, PA-D-L.

 
Secondary Structure of the Lipopeptides in the Membrane
The secondary structure of the lipopeptides was measured in PC/PE/PI/ergosterol multibilayers by using ATR-FTIR spectroscopy. We exchanged hydrogen with deuterium (see "Experimental Procedures") to make it possible to differentiate {alpha}-helical components from a random structure, since the absorption of the random structure shifts to a greater extent than the {alpha}-helical component after deuteration (36). The amide I region spectra of the bound lipopeptides are shown in Fig. 3. Second derivatives accompanied with Savitsky-Golay smoothing were calculated to identify the positions of the component bands in the spectra and are given in the corresponding panels in Fig. 3. We used these wave numbers as initial parameters for curve fitting with gaussian component peaks. Table V summarizes the assignments, wave numbers ({nu}), and relative areas of the component peaks. The assignment of the different secondary structures to the various amide I regions was calculated according to the values taken from Jackson and Mantsch (37). The data reveal that the major amide I band of PA-D-V is located at ~1627 cm–1 (78 ± 1%), corresponding to the aggregated {beta}-sheet, whereas that of PA-D-L has two peaks at 1635 cm–1 (44%) and 1652 cm–1 (32%), suggesting the existence of both {beta}-sheet and {alpha}-helical structures. In contrast, both PA-D-G and PA-D-A are predominantly random coils. CD spectroscopy in lysophosphatidylcholine confirmed these findings (Fig. 4).



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  		FIG. 3.
FTIR spectra of the fully deuterated amide I band (1600–1700 cm–1) and the corresponding second derivative of the lipopeptides in PC/PE/PI/ergosterol (5:2.5:2.5:1 w/w) multibilayers. The sum of the fitted components (thick line) and the fitted components (thin line) of the lipopeptides are presented in the upper panel of each scheme. Second derivatives were calculated to identify the positions of the component bands in the spectra and are presented in the lower panel of each scheme. Panel A, PA-D-G. Panel B, PA-D-A. Panel C, PA-D-V. Panel D, PA-D-L. All curves represent the experimental FTIR spectra after Savitzky-Golay smoothing. A 60:1 lipid/diastereomer molar ratio was used.

 


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  		TABLE V
Secondary structure of the lipopeptides in PC/PE/PI/ergosterol (5:2.5:2.5:1 w/w) multibilayer

The results are the average of four independent experiments. A 1:60 peptide:lipid molar ratio was used.

 



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  		FIG. 4.
CD spectra of the lipopeptides in lysophosphatidylcholine micelles. Spectra were taken at a lipopeptide concentration of 100 µM in double-distilled water containing 1% of lysophosphatidylcholine. The assay was performed as described under "Experimental Procedures." The designations are as follows: {triangleup}, PA-D-G; {square}, PA-D-A; {blacktriangleup}, PA-D-V; {blacksquare}, PA-D-L.

 
Orientation of the Phospholipid Membrane and the Effect of the Lipopeptides on the Phospholipid Acyl Chain Order
Polarized ATR-FTIR spectroscopy was used to determine the orientation of the lipid membrane. The symmetric [vsym(CH2) {approx} 2853 cm–1] and the antisymmetric [vsym(CH2) {approx} 2922 cm–1] vibrations of lipid methylene C-H bonds are perpendicular to the molecular axis of a fully extended hydrocarbon chain. Thus, measurements of the dichroism of infrared light absorbance can reveal the order and orientation of the membrane sample relative to the prism surface. However, because the intensity of the antisymmetric CH2 vibration was higher than 1, our calculations were based only on R values taken from the symmetric vibration. The data revealed that the membranes are predominantly in a liquid crystalline phase (r = 1.33), indicating that the lipid multibilayers used were well oriented (29, 38). The effect of the lipopeptides on the multibilayer acyl chain order can be estimated by comparing the CH2-stretching dichroic ratio of pure phospholipid multibilayers and membrane-bound lipopeptides. The data indicate that the highest effect on the lipid order was obtained with PA-D-V (R = 1.55), whereas the incorporation of all the other lipopeptides into the membrane changed the lipid order to a much lesser extent (R = 1.44), suggesting they are surface-localized.

In Vivo Transmembrane Potential Depolarization
Transmembrane Potential Depolarization on the Yeast C. neoformans—To test the ability of the lipopeptides to permeate the microorganism cell wall, we monitored the collapse of the transmembrane potential in living yeast in the presence of the lipopeptides by using a potential-sensitive dye. All the lipopeptides were able to dissipate the membrane potential of C. neoformans to a high extent (Fig. 5, panel A). Interestingly, these results correlate well with the antifungal activity of the different peptides against C. neoformans. However, there is a slight difference between the MIC and the concentration of peptides required to dissipate 100% of the yeast transmembrane potential. This might be due to differences in the conditions used for the antifungal assay and the potential depolarization assay. These results suggest that the target of the lipopeptides is the plasma membrane of the cells.



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  		FIG. 5.
Maximal dissipation of the diffusion potential induced by the lipopeptides by using intact yeast and E. coli D21 spheroplasts. The lipopeptides were added to C. neoformans conidia (panel A) or E. coli D21 spheroplasts (panel B) that were preincubated with the fluorescent dye diS-C3-5 for 5 min. Membrane depolarization was monitored by an increase in the fluorescence of diS-C3-5 (excitation wavelength {lambda}ex = 622 nm, emission wavelength {lambda}em = 670 nm) after the addition of peptides at different concentrations. Fluorescence recovery was measured as a function of time with 5–10 min of intervals, and its maxima were reported. {triangleup}, PA-D-G; {square}, PA-D-A; {blacktriangleup}, PA-D-V; {blacksquare}, PA-D-L.

 
Transmembrane Potential Depolarization of E. coli Spheroplasts—Because the cytoplasmic membrane of bacteria is negatively charged compared with the zwitterionic membrane of fungi, we performed potential depolarization assays using spheroplasts of the Gram-negative bacteria E. coli D21. A dose-response curve of the lipopeptide-mediated potential depolarization activity is shown in Fig. 5, panel B. All the lipopeptides are highly active in disrupting the membrane potential of the E. coli D21 strain. However, a good correlation between the antibacterial activity and the potential to permeate the cytoplasmic membrane was found only with PA-D-G and PA-D-A. The findings that both PA-D-V and PA-D-L are highly active on spheroplasts but not on intact bacteria and that both form large oligomers may indicate that they are not active on E. coli because they are not able to penetrate through the cell wall into the cytoplasmic target membrane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The most interesting result in this study is the finding that the attachment of palmitic acid to the N terminus of positively charged short peptides, which are inert toward microorganisms (D-G, D-A peptides), endowed them with a broad spectrum of potent antibacterial and antifungal activity and with a low hemolytic activity against the highly diluted solution of hRBCs. In contrast, the same modification to a potent antimicrobial peptide (D-L peptide) abolished its antibacterial activity but increased both its antifungal and hemolytic activities. Furthermore, the parental D-V peptide was inactive toward bacteria, fungi, and yeasts, whereas its palmitoylated form was active only toward the two strains of yeasts tested (Tables II and III).

Many studies with membrane-active peptides have demonstrated the important role of hydrophobicity and structure for their biological function (39, 40). Here all four parental peptides were derived from a similar peptidic backbone and contain the same number of positive charges located at the same positions. However, they differ markedly in their hydrophobicities. We show that palmitic acid attached to the N terminus of the inactive peptides can compensate for the hydrophobicity of the peptidic chain. Note that shorter acyl analogues could enhance the antibacterial activity of a peptide fragment of human lactoferrin, which originally had weak antibacterial activity (41), or endowed antibacterial peptides with antifungal activity (21).

The Peptidic Moieties Have Different Structures and Organizations within the Lipopeptide Oligomers—To shed light on the mode of action of these new lipopeptides, we examined their oligomeric state and their ability to increase the membrane permeability as well as their structure and location when bound to membranes. Previous studies showed that the parental non-palymitoylated peptides differ markedly (i) in their ability to bind and permeate phospholipid membranes, (ii) in their structure in the membrane, and (iii) in the oligomeric state in solution and membranes (22). These studies show that D-G and D-A peptides are inert toward both zwitterionic and negatively charged phospholipid membranes and are monomeric in solution. In contrast, both D-V and D-L peptides bound strongly and permeated both types of phospholipid membranes. However, whereas D-V forms oligomers in solutions and membranes, D-L is monomeric. In addition, all the parental peptides were also found to be unstructured in solution.

Here, all the palmitoylated peptides are expected to form micelles. Indeed, previous studies showed that the micellar concentration of palmitic acid conjugated to an amino acid is in the µM range and should significantly decrease by increasing the peptidic chain (42). However, the structure and the organization of the peptidic chains in solution are different for the lipopeptides. CD spectroscopy revealed a defined {alpha}-helical structure in solution only for PA-D-L (Fig. 2). Furthermore, the Trp environment of this peptide is more hydrophobic than that expected in solution (Table IV). This may happen if the peptidic chain interacts with the micelles formed by the palmitic moiety. Regarding PA-D-V, the finding of strong quenching of the Trp supports the notion that in addition to the micelles formed by the palmitic group, the peptidic chain of PA-D-V is also aggregated. In contrast, the relatively hydrophilic environment of the Trp in PA-D-G and PA-D-A suggests that their peptidic moiety is not aggregated and not intercalated within the palmitic micelles. This in agreement with the findings that these two parental peptides bind only weekly to lipids (22).

The Role of Lipopeptide Oligomerization on Target-cell Specificity—Previous studies show that the peptide oligomerization is a characteristic of antimicrobial peptides that act on zwitterionic phospholipids, which are abundant in the plasma membrane of variable eukaryotic organisms from fungi to humans (18, 19, 43). Because of this, such peptides have both antifungal and hemolytic activities. However, whereas reversible oligomerization has been shown to be important for antibacterial and antifungal activity, it was not crucial for hemolytic activity because covalently linked pentamers of antimicrobial peptides became highly hemolytic (44). Here all four lipopeptides form oligomers in solution. However, in two of them, the Val and Leu lipopeptides, the peptidic chains stabilize the oligomers; in PA-D-V there is peptide-peptide interaction, and in PA-D-L, there is peptide-palmitoyl interaction. Therefore, the dissociation of these two oligomers is probably more difficult than the dissociation of the lipopeptides containing Gly and Ala once they bound to the target cell.

Oligomer dissociation seems to be an important requirement for antimicrobial activity because many pathogens including bacteria, yeasts, and fungi are surrounded, in addition to the plasma membrane, by an external barrier, which contains mainly polysaccharide compounds. Therefore, to reach the cytoplasmic phospholipid membranes (the possible target of the lipopeptides, as will be discussed in the following paragraph), they need to traverse the microorganism cell wall. As soon as the peptides cross the cell wall of the pathogen, the only barrier left is the plasma membrane. Thus, the smaller the size of the molecule, the higher is its ability to penetrate through the cell wall to reach the plasma membrane. This should result in improved biological activity. Hancock and co-workers termed this process for Gram-negative bacteria a self-promoted uptake (45). Indeed, the most active and those with the broadest spectrum of activity are PA-D-G and PA-D-A. However, both are also the less hemolytic peptides. This can be explained if we take into account the negatively charged glycocalix layer surrounding the cytoplasmic membrane of erythrocytes. The positively charged peptides are probably attached first to the glycocalix layer via electrostatic interaction. However, because the peptidic moiety has low affinity to zwitterionic membranes, it makes it more difficult for these two peptides to partition into the cell phospholipid membrane compared with the more hydrophobic ones.

The Plasma Membrane Is the Main Target of the Lipopeptides—Similar to other antimicrobial peptides, the main target of the lipopeptides is the plasma membrane. This is supported by the finding of a correlation between the antifungal activity of the lipopeptides against C. neoformans and their ability to disrupt the membrane potential of the intact yeast (Fig. 5, panel A). In addition, when we removed the cell wall of E. coli (by preparing spheroplasts), all the lipopeptides possessed high cell-permeating activity (Fig. 5, panel B), which correlates with the hydrophobicities of the peptides. We have also performed studies by using model phospholipid membranes. These studies included (i) measuring the environment of the Trp residue when bound to vesicles, (ii) determining the effect of the four lipopeptides on the acyl chain order, and (iii) determining the secondary structure of the lipopeptides within the phospholipid membranes. The data revealed that three lipopeptides PA-D-G, PA-D-A, and PA-D-L, had a strong and similar effect on the lipid order as well as the extent of blue shift of the Trp. This is true despite differences in their secondary structures. However, PA-D-V was an exception, because it disrupted the lipid order to a greater extent, and no fluorescence signal could be detected from its Trp. This is probably because the peptide is aggregated and the Trp fluorescence is fully quenched. This was confirmed by ATR-FTIR spectroscopy which showed that PA-D-V forms mostly {beta}-aggregates in the membrane, whereas PA-D-L adopted a predominantly helix/dynamic helix, and PA-D-G and PA-D-A adopted random coils.

Note that native lipopeptides already exist, and they possess a broad spectrum of activities, including antibacterial, antifungal, antiviral, and cytolytic activities (46–50) (for review see Ref. 51). However, compared with the lipopeptides presented here, most of them have a unique alkyl chain bound to a short peptide chain (6–7 amino acids) and are cyclic (52) and composed mainly of hydrophobic and acidic L- and D-amino acids. They have been shown to act either via nonspecific membrane lysis, e.g. iturin (53) and surfactin (54), or via inhibition of the synthesis of cell wall components, e.g. the echinocandins family, which inhibits the synthesis of 1,3-{beta}-D-glucan (55) (for review see Ref. 9). However, although the echinocandins, especially caspofungin, were approved by the Food and Drug Administration for therapeutic usage (56, 57) they are limited mostly to Candida and Aspergillus species, whereas the cryptococcal disease remains uncovered by these drugs. Furthermore, their ability to act on a specific target makes them more amendable to develop resistance. The biophysical studies presented here together with the findings of a direct correlation between the MIC and the in vitro and in vivo transmembrane potential depolarization assays suggest that our lipopeptides act directly on the cell plasma membrane and not on a specific target. Furthermore, previous studies revealed that the position of the D-amino acids of a diastereomeric peptide can be changed to produce a repertoire of new active compounds that differ from each other by their structure in solution (and, hence, should have different antigenicities) and by their susceptibility to enzymatic degradation (58). Controlled enzymatic degradation is an advantage because lytic peptides that are fully degraded or fully protected from enzymatic degradation are less suitable for therapy. Further studies on this new family of lipopeptides, which are highly active toward both bacteria and C. neoformans, should assist in the development of broad-spectrum antifungal agents.


   FOOTNOTES
 
* This study was supported by the Israel Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} The Harold S. and Harriet B. Brady Professorial Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-9342711; Fax: 972-8-9344112; E-mail: Yechiel.Shai{at}weizmann.ac.il.

1 The abbreviations used are: PC, phosphatidylcholine; Fmoc, fluorenylmethoxycarbonyl; ATR-FTIR, attenuated total reflectance Fouriertransform infrared; CD, circular dichroism; hRBC, human red blood cells; PBS, phosphate buffered saline; PI, phosphatidylinositol; PE, phosphatidylethanolamine; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; MIC, minimal inhibitory concentration; diS-C3-5, 3-3'-dipropylthiadicarbocyanine iodide; PA, palmitoylated. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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