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Originally published In Press as doi:10.1074/jbc.M610555200 on May 7, 2007

J. Biol. Chem., Vol. 282, Issue 26, 18831-18841, June 29, 2007
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Energy Depletion Protects Candida albicans against Antimicrobial Peptides by Rigidifying Its Cell Membrane*

Enno C. I. Veerman{ddagger}1, Marianne Valentijn-Benz{ddagger}, Kamran Nazmi{ddagger}, Anita L. A. Ruissen{ddagger}, Els Walgreen-Weterings{ddagger}, Jan van Marle§, Alexander B. Doust, Wim van't Hof{ddagger}, Jan G. M. Bolscher{ddagger}, and Arie V. Nieuw Amerongen{ddagger}

From the {ddagger}Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Vrije Universiteit and Universiteit van Amsterdam, 1081 BT Amsterdam, The Netherlands, the §Department of Cell Biology and Histology, Central Electron Microscopy, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands, and the Department of Physics and Astronomy, Biophysics Section, Vrije Universiteit, 1105 AZ Amsterdam, The Netherlands

Received for publication, November 14, 2006 , and in revised form, April 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me2SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the last few decades an expanding number of antimicrobial peptides have been isolated from virtually all classes of organisms, where they play an important role in the innate defense against microbial and viral infections. Characterization of these peptides has revealed a wide diversity in amino acid sequences, yet they share characteristic features; they are usually polycationic and amphipathic, containing both a hydrophilic and a hydrophobic side. This promotes their insertion into and transmigration over the cytoplasmic membrane of the target cell, with killing of the cell as a final consequence. Interestingly, cellular sensitivity to cationic proteins and peptides such as salivary histatins and defensins is diminished by conditions that affect the energy status of the target cell (1–6). This effect is not restricted to cationic peptides, because azoles are also sensitive to the energy status of Candida glabrata (7). In Chlorella metabolic inhibition abolishes the membrane-disruptive effects of the polyene nystatin and even of the detergent Triton X-100 (8). As explanation for the desensitizing effects of energy depletion, it has been proposed that interaction of cationic peptides with the target cell would involve active transport systems, which for their activity are dependent on the energy charge of the cell (1, 9–12). However, direct experimental proof corroborating this hypothesis is still lacking.

The present study addresses the question of how on a molecular level the energy metabolism is linked with the sensitivity of the Candida cell to antimicrobial peptides. Thus far, many different types of mechanisms have been identified that contribute to an acquired drug-resistant phenotype in yeast cells, including the overexpression of energy-driven efflux pumps, mutations in the target enzyme, or alterations in biosynthetic pathways (13). The resistance induced by energy depletion seems fundamentally different from these mechanisms, because it is a direct response to a physiological stress condition, which protects the yeast against a range of natural and pharmacological anti-fungal agents. Thus, elucidation of this molecular resistance mechanism not only will deepen our insight in the working mechanism of antimicrobial peptides but will aid in the development of (non)peptide therapeutics that are suited for fighting infections under energy-restricted conditions, such as in biofilms.

The Histatin 5 (Hst5)2-mediated killing of Candida albicans is used in the present study as a model system to comprehensively investigate the different aspects of the peptide-target cell interaction, including the role of the cell wall, the membrane potential, and the physical state of the membrane, in relation to the energy charge of the cell. We found that energy depletion induced a decrease in membrane fluidity, which was reversed by the membrane fluidizer benzyl alcohol. In line, the increased resistance of energy-depleted C. albicans against Hst5 was reversed by the membrane fluidizer benzyl alcohol, without restoration of the energy charge of the cell. On the other hand, inducing increased membrane rigidity by lowering the temperature or by treatment with a membrane-rigidifying agent led to an increased resistance against Hst5. It is hypothesized that the actin cytoskeleton, which is highly sensitive to the energy charge of the cell mediates the effect, because the cytoskeleton inhibitor jasplakinolide also induced resistance to Hst5.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and FITC Labeling of the Peptides—Peptides (see Table 1) were manufactured by solid phase peptide synthesis using Fmoc chemistry with a MilliGen 9050 peptide synthesizer (Milligen-Biosearch, Bedford, MA) according to the manufacturer's procedures. N-{alpha}-Fmoc protected amino acids and preloaded polyethylene glycol-polystyrene supports were obtained from Applied Biosystems (Foster City, CA). The peptides were purified by reverse phase high pressure liquid chromatography (Jasco Corporation, Tokyo, Japan) to a purity of at least 90%, and the authenticity of the peptides was confirmed by ion trap mass spectrometry with a LCQ Deca XP (Thermo Finnigan, San Jose, CA). FITC labeling of Hst5 and Dhvar5 was performed as described previously (3). The labeled peptides were designated F-Hst5 and F-Dhvar5, respectively. Both in the viability assay and in the propidium iodide assay, fluoresceinlabeled peptides exhibited comparable activities against C. albicans as the parent peptides.


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  		TABLE 1
The effect of energy depletion on the activity of various antimicrobial peptides

C. albicans were preincubated with PPB with NaCl or PBB with NaN3 for 15 min and then incubated with 2-fold serially diluted peptides in the same buffers for 1 h. After appropriate dilution, the aliquots (in duplicate) were plated on Sabouraud dextrose agar plates. After 2 days the numbers of colony forming units was determined. LC50 indicates the concentration at which 50% of the cells were killed.

 
Growth Conditions—C. albicans (ATCC 10231) cultured aerobically at 30 °C on Sabouraud dextrose agar plates (Oxoid, Hampshire, UK) was suspended in 25 ml of Sabouraud dextrose broth in a 100-ml Erlenmeyer flask. After 20 h of incubation at 30 °C, 1 ml from this suspension was subcultured for 1–2 h in 20 ml of Sabouraud dextrose broth to obtain a mid-log phase culture. The cells were washed twice in 1 mM potassium phosphate (PPB) and resuspended to a cell density of 2 McFarland (~107 cells/ml).

Spheroplast Preparation—One gram (wet weight) of C. albicans of a mid-log phase culture was suspended in 100 mM Tris buffer (pH 7.4), supplemented with 100 mM EDTA (TE buffer) and incubated for 45 min with beta-mercaptoethanol. The cells were washed and suspended in 4 ml of TE buffer, supplemented with 1 M sorbitol, and incubated with 100 µl zymolase (50 units). After treatment, more than 90% of the cells were converted to spheroplasts, as determined by counting cells after lysis in distilled water. The spheroplasts were resuspended in PPB supplemented with 0.5 M sorbitol as osmoprotectant.

Determination of the Membrane-disruptive Activity of Peptides (PI Assay)—Membrane-disruptive activity of peptides was determined by monitoring the fluorescence enhancement of propidium iodide (Invitrogen) in peptide-treated cells, as described previously (14). The membrane impermeant PI only enters membrane-compromised cells, after which the fluorescence of this probe is enhanced by 20–30-fold because of its binding to nucleic acids. Cell suspensions were mixed with the indicated agents and incubated at 30 °C for 30 min (unless indicated otherwise) with gentle shaking. NaN3 was dissolved in PPB; cyanide m-chlorophenylhydrazone (Sigma-Aldrich) was dissolved in methanol; and rapamycin (Sigma-Aldrich), valinomycin (Sigma-Aldrich), latrunculin B (MP Biomedicals), and jasplakinolide (Invitrogen) were dissolved in Me2SO and diluted prior to use. The final concentrations of methanol and Me2SO did not exceed 0.1%. Controls were incubated with PPB supplemented with appropriate volumes of the corresponding vehicle (Me2SO or methanol) alone. Cell suspensions were supplemented with PI (final concentration, 10 µM) and subsequently added to 2-fold serial dilutions of peptides. PI fluorescence was measured at 5-min intervals for 1 h, at excitation and emission wavelengths of 544 and 620 nm, respectively, in a Fluostar Galaxy microplate fluorimeter (BMFG Labtechnologies, Offenburg, Germany). Afterward, the numbers of surviving cells were determined by plating aliquots on Sabouraud dextrose agar plates and counting colony-forming units, as described below.

Candidacidal Activity of Peptides—The effects of the various agents and treatments on the viability of C. albicans were determined in a microdilution viability assay, essentially as described previously (3). In brief, 50-µl aliquots from a mid-log phase culture of C. albicans were incubated with equal volumes of peptide solutions (0.3–100 µM) and incubated at 30 °C for 60 min. The incubation mixtures were appropriately diluted (200–500-fold) in phosphate-buffered saline, and 25-µl aliquots were plated on Sabouraud dextrose agar plates. After 48 h of incubation at 30 °C, the numbers of colony-forming units were counted.

DiSC3(5) Fluorescence to Monitor Membrane Potential—Induction of release of the fluorescent probe DiSC3(5) (Invitrogen) was monitored to study the effect of azide on the membrane potential of C. albicans (15). C. albicans (~107 cells/ml) in PPB were incubated with a final concentration of 1.6 µM DiSC3(5) at 30 °C until a constant fluorescence level was achieved (after approximately 10 min). Next either 5 mM NaN3 or 5 mM NaCl (control experiment) was added, followed by an excess of Hst5. An excess of Dhvar4 (8 times its LC50 value) was added to achieve complete dissipation of the cytoplasmic membrane potential. Changes in peptide-mediated fluorescence intensity were monitored with a Perkin-Elmer LS 50 B spectrofluorimeter (Perkin-Elmer).

FACS Analysis—FACS analysis was performed essentially as described previously (3). C. albicans (~107 cells/ml) were preincubated in PPB with 5 mM NaN3 at 30 °C for 15 min. Subsequently, equipotent concentrations of FITC-labeled peptides were added (65.5 µM F-Hst5 or 17 µM F-Dhvar5), and incubation was continued for another 15 min. The cells were washed twice with PPB with NaN3, suspended to a density of ~107 cells/ml, and diluted 10-fold in the same buffer before use. Cell-associated fluorescence was measured with a FACS apparatus (Becton Dickinson, Franklin Lakes, NJ) using a 15 mM argon laser at 488 nm for excitation and a 530-nm filter for detection of emitted light. In the control experiment, cells the were treated the same way, except that NaN3 was replaced by NaCl.

Confocal Microscopy—C. albicans (~107 cells/ml) were preincubated with PPB with NaN3 at 37 °C for 15 min. FITC-labeled peptides were added (final F-Hst5 concentration, 65 µM; final F-Dhvar5 concentration, 17 µM), and incubation was continued for another 15 min. To remove unbound peptide, the cells were washed with PPB with NaN3, centrifuged (5 min at 10,000 x g), and resuspended in the same buffer. The resulting suspensions were divided into two equal volumes, which were centrifuged (5 min at 10,000 x g) and of which one pellet was resuspended in PPB with NaN3, the other in PPB with NaCl. Cells treated with Hst5 or Dhvar5 in PPB with NaCl served as positive controls. The cells were examined with a Leica TCS NT confocal system (Leica Microsystems, Heidelberg, Germany) equipped with an argon/krypton laser and a 100x NA 1.4 object lens.

Electron Microscopy—Ultrastructural localization was examined using immunogold-labeling and transmission electron microscopy as described previously (6). C. albicans (~107 cells/ml) were preincubated with either PPB with NaN3 or PPB with NaCl and incubated with 65.5 µM F-Hst5, or 17 µM F-Dhvar5 at 37 °C for 15 min. Fixation, preparation of cryo-sections, and incubation with gold-labeled mouse anti-FITC antibodies (Aurion, Wageningen, The Netherlands) were performed as described previously (6). The cells were examined using a Philips EM-420 transmission electron microscope (Philips, Eindhoven, The Netherlands).

Determination of the Adenine Nucleotide Content of C. albicans—Cellular content of adenine nucleotides was determined as previously described (14). In short, C. albicans (~107 cells/ml) were suspended in PPB supplemented with 5 µg/ml guanosine bromide as internal standard. After treatment with various agents or incubation under various conditions, 400 µl of the cell suspension (in duplicate) was transferred to 80% boiling ethanol, buffered with 50 mM NH4HCO3 (pH 7.8) (16) to lyse the cells, and boiled for 3 min. After freeze-drying, the resulting pellet was resuspended in 100 µl of distilled water and centrifuged (10 min at 10,000 x g). Aliquots of the supernatants were analyzed by capillary zone electrophoresis with a BioFocus 2000 capillary electrophoresis system (Bio-Rad), equipped with an uncoated fused-silica capillary (internal diameter, 50 µM; length, 50 cm).

Kinetics of azide-induced effects on the adenine nucleotide composition were determined in the same way using NaN3 (final concentration, 5 mM) in the incubation buffer. At different time intervals 400-µl aliquots were taken and transferred into buffered boiling ethanol and processed as described above. The control incubations were carried out in the same way, except that NaCl was added instead of NaN3.

To monitor the reversibility of the effect of azide on the intracellular adenine nucleotide composition, the cells were incubated with 5 mM NaN3 for 30 min. Then 400-µl aliquots (in duplicate) were taken and processed as described above. Subsequently, NaN3 was removed by washing twice in 9 ml of PPB. From the resulting suspension 400-µl aliquots (in duplicate) were taken at different time intervals and processed as described above for measurement of the adenine nucleotide content in comparison with the initial content.

Fluorescence Anisotropy of DPH—The effect of energy poisons on the physical state of the yeast plasma membrane was measured in whole cells using the membrane fluidity probe DPH as described previously (17, 18). In short, a mid-log phase culture of C. albicans was washed twice and then suspended in PPB to a density of 8 mg/ml wet weight. The cells were incubated at 20 °C for 5 min, and the DPH was added to a final concentration of 2 µM. The same volume of the solvent (Me2SO) was added to the control cells. Incubation was continued for 20 min at 20 °C, after which the cells were washed twice with PPB. Subsequently the DPH-treated cells were suspended in the same volume of PPB supplemented with 5 mM NaN3, 5 mM NaN3 with 50 mM benzyl alcohol, PPB with 50 mM benzyl alcohol, and PPB with 5% (v/v) Me2SO, respectively. Immediately thereafter, fluorescence emission anisotropy measurements were performed in a 1-cm quartz cuvette on a Fluoromax-3 fluorimeter (Horiba Jobin Yvon, Longjumeau, France) in the right angle conformation. The optical densities of the samples were kept low to avoid inner filter effects. The samples were stirred continuously throughout the measurements. Upon 365-nm excitation, emission wavelengths were scanned from 422 to 432 nm using emission and excitation slits of 4.5 nm. In the 422–432-nm wavelength range, the value for fluorescence anisotropy of DPH is constant (19). The temperature of the samples was maintained at 30 or 4 °C using a thermostatted water bath. All four combinations of vertically and horizontally polarized excitation and emission light were measured, and the fluorescence anisotropy (r) was automatically calculated. Anisotropy values of three repeated measurements of various conditions were statistically analyzed by one-way analysis of variance, followed by post hoc multicomparison with Tukey test. The data were analyzed with Statistical Package for the Social Sciences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Energy Metabolism on Candidacidal Activities of Antimicrobial Peptides—In previous studies we found that Hst5 accumulates in the mitochondria of C. albicans (3, 20). Because treatment with mitochondrial poisons such as sodium azide renders C. albicans cells insensitive to Hst5 (3, 20), it was tempting to speculate about a causative relationship between the cellular target of the peptide and the desensitizing effects of azide. Therefore, we addressed the issue of whether the effects of azide and other energy poisons were specific for Hst5 and tested a number of structurally different peptides for their sensitivity to azide: (i) Hst5 and Hst5-derived peptides, including dh5 (residues 11–24), Dhvar4 (a multi-substituted variant of dh5), P-113 (encompassing residues 4–15 of Hst5), and its D-enantiomer P-113D; (ii) the bovine lactoferrin-derived peptides (21) LFcin 17–30 and LFampin 265–284; (iii) drosocin and a drosocin variant (22); and (iv) the artificial peptide Dhvar5. The peptides were tested in a viability assay as well as in a microtiter plate assay using PI to monitor their membranolytic effects. The membrane-impermeant PI only enters membrane-compromised cells, after which the fluorescence of this probe is enhanced by 20–30-fold because of its binding to nucleic acids. The candidacidal activities (Table 1) and the membranolytic activities of all peptides tested were inhibited in the presence of azide. Differences in sensitivity to azide, however, were noted: Hst5, dh5, both P-113 and its enantiomer P-113D, LFcin 17–30, and the drosocin peptides were completely inactive in the presence of azide. On the other hand, Dhvar5 and LFampin 265–284 were intermediately active, whereas the activity of Dhvar4 was marginally diminished (Table 1). These results were mirrored in the membranolytic assays with PI (not shown). This was in line with previous results showing that peptide-mediated PI uptake is accompanied by leakage of vital cell constituents, including ATP and other adenine nucleotides, resulting in cell death (14).

We decided to use the extremely azide-sensitive Hst5 as a model peptide to explore the molecular basis underlying the apparent correlation between the energy metabolism of the yeast cell and its sensitivity for antimicrobial peptides. In parallel, the effect on the intracellular adenine nucleotide content was systematically monitored. The addition of azide virtually instantaneously inhibited Hst5-mediated influx of PI (Fig. 1A), and concomitantly depleted the energy charge of the cell (Fig. 2A). These effects were readily reversed after washing out of the energy poison (Figs. 1B and 2B). This illustrated that the sensitivity of the cell to Hst5 was closely linked with its energy charge. Other energy poisons such as the potassium ionophore valinomycin and the protonophore CCCP likewise depleted the energy charge of the cell and blocked the Hst5-mediated PI influx (Figs. 1C and 2C). The inhibitory effects of these energy poisons were also confirmed in viability assays (not shown).

Azide Reversibly Inhibits the Internalization of Hst5 in C. albicans—Several stages can be distinguished in the candidacidal process. It has been proposed that in the first stage, Hst5 associates transiently with a putative receptor at the cell wall (23). This is followed by transmigration over the membrane and accumulation in the cell. To identify the step in the killing process that is sensitive to the energy charge of the yeast cell, we examined the effect of azide on the association between F-Hst5 and C. albicans by FACScan analysis (Fig. 3). Introduction of the fluorescein group marginally influences the candidacidal and membrane disrupting activity of the peptides (Table 1). To compensate for ionic strength effects caused by the presence of sodium and azide ions, the incubations without sodium azide were carried out in PPB supplemented with an equimolar concentration of sodium chloride. After incubation with F-Hst5 in the absence of azide, the cell-associated fluorescence increased to a value that was a thousand times higher than that of control cells, which had been incubated with either a fluoresceintagged irrelevant peptide or without any peptide. The cells treated with F-Hst5 in PPB with NaN3 exhibited a 100-fold lower fluorescence, but this was still ~10-fold higher than that of the negative control. This fluorescence remained after repeated washing of the cells with PPB with NaN3. For comparison we examined the effect of energy depletion on the association between Dhvar5, which was moderately sensitive to azide and C. albicans. In this case the F-Dhvar5 fluorescence was decreased in about 50% of cells, whereas the rest of the cells remained intensely labeled (Fig. 3B).


Figure 1
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  		FIGURE 1.
The effect of energy poisons on Histatin 5-mediated influx of propidium iodide in C. albicans. A, C. albicans was incubated with PPB with 5 mM NaCl and directly transferred to equal volumes of 20 µM Hst5 in PPB with NaCl ({blacksquare}) or PPB with NaN3 ({diamondsuit}) supplemented with PI. After mixing, the development of fluorescence was monitored at 100-s intervals at 30 °C at {lambda}ex 544 nm and {lambda}em 620 nm. B, suspensions of C. albicans (107 cells/ml PPB) were mixed with either NaN3 or NaCl (final concentration, 5 mM) at 30 °C for 30 min. To remove azide, the cells were washed twice in PPB with NaCl. As control served cells that were washed in PPB with NaN3. Immediately after washing, the cells were transferred to equal volumes of 20 µM Hst5 in the indicated buffer, supplemented with PI. The development of fluorescence was monitored at 30-s intervals for 1 h at 30 °C at {lambda}ex 544 nm and {lambda}em 620 nm. {blacksquare}, cells preincubated in PPB with NaCl and then resuspended in PPB with NaCl. {diamondsuit}, cells preincubated in PPB with NaN3 and then resuspended in PPB with NaCl. {diamondsuit}, cells preincubated in PPB with NaN3 and then resuspended in PPB with NaN3. The curves are representative of two independent experiments, carried out in duplicate. C, suspensions of C. albicans (2 x 107 cells/ml) were incubated with PPB supplemented with 5 mM NaCl ({diamondsuit}), 5 mM NaN3 ({blacksquare}), 10 µM valinomycin (Val, {blacktriangleup}), or 2.5 µM CCCP (•) for 30 min. PI was added, and the cells were transferred to wells containing equal volumes of serially diluted Hst5 in PPB supplemented with the corresponding agent in the same concentration. Development of fluorescence was followed for each concentration of the peptides at 5-min intervals for 1 h at {lambda}ex 544 nm and {lambda}em 620 nm. The figure shows the fluorescence, expressed in arbitrary units (AU), after 1 h of incubation. The curves are representative of three independent experiments, carried out in duplicate.

 


Figure 2
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  		FIGURE 2.
The effect of energy poisons on the energy charge of C. albicans. A, C. albicans (2 x 107 cells/ml PPB) was mixed with 5 mM NaN3. At the indicated time points, aliquots were taken and transferred into boiling ethanol to lyse the cells. Adenine nucleotide content was determined by capillary zone electrophoresis (CZE). {diamondsuit}, ATP; {blacktriangleup}, AMP. In control cells treated with NaCl, no measurable decrease in ATP occurred during the time course of the experiment (not shown). B, 10-ml suspension of C. albicans (2 x 107 cells/ml) was incubated in PPB supplemented with 5 mM NaN3 at 30 °C. After 5 min a 400-µl aliquot was taken and boiled in buffered ethanol. The remaining cells were washed two times in PPB to remove NaN3 and resuspended in 9 ml PPB. At the indicated time points, 400-µl aliquots were taken and processed for determination of the intracellular adenine nucleotide content. {diamondsuit}, ATP; {blacktriangleup}, AMP. The curves are representative of two independent experiments, carried out in duplicate. C, C. albicans was incubated in PPB supplemented with 5 mM NaCl, 10 µM valinomycin (Val), 2.5 µM CCCP, and 5 mM NaN3, respectively. After 30 min of incubation, the cells were lysed by transfer into boiling ethanol. Adenine nucleotide content was determined by CZE. Black bars, ATP; shaded bars, ADP; white bars, AMP. No measurable amounts of adenine nucleotide were released in the supernatant of intact untreated cells in the time course of these experiments. The data are representative of at least two independent experiments, carried out in duplicate. The values represent the means of duplicate measurements.

 


Figure 3
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  		FIGURE 3.
The effect of energy depletion on the binding of Histatin 5 and Dhvar5 to C. albicans, analyzed with FACScan. Suspensions of C. albicans (3.2 x 106 cells/ml) were preincubated with PPB (at 37 °C for 15 min) supplemented either with 5 mM NaCl or with NaN3. Subsequently, F-Hst5 (A) or F-Dhvar5 (B) was added, and incubation was continued for an additional 15 min. A, in the presence of NaN3, binding of F-Hst5 to C. albicans was ~100-fold lower than in the presence of NaCl. The fluorescence signal of cells incubated with F-Hst5 in the presence of NaN3 was still ~10-fold higher than that of cells that had been incubated with an irrelevant fluoresceinated peptide, F-CystS, (control). B, binding of Dhvar5 to C. albicans was ~100-fold diminished in ~50% of the cells. Also in this case the residual fluorescence was ~10-fold higher than that of the negative control.

 
From Fig. 3 no distinction can be made between surface-bound and internalized peptides. To obtain information on the location of the peptides, fluorescence microscopy was conducted on cells treated with F-Hst5 in the absence and presence of azide (Fig. 4). In the absence of azide F-Hst5 accumulated inside the cells, producing a granular labeling pattern, whereas the cell boundaries were negative (Fig. 4A). Upon depletion of the ATP charge by azide, in virtually all cells the cytoplasmic labeling was absent. In 5–10% of the cells a faint peripheral labeling could be observed (Fig. 4B). After subsequent removal of azide, which restored the energy charge of the cells, the intracellular labeling pattern returned (Fig. 4C). This suggests that the peripheral labeling in the presence of azide represented a transient stage that under normal conditions is not detected.


Figure 4
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  		FIGURE 4.
Confocal fluorescence microscopy of C. albicans after incubation with F-Histatin 5. Left column, suspensions of C. albicans (3.2x106 cells/ml) were preincubated at 37 °C for 15 min in PPB with NaCl or PPB with NaN3. Then F-Hst5 was added and incubation was continued for 15 min. A, C. albicans treated with F-Hst5 in PPB with NaCl. B, C. albicans treated with F-Hst5 in PPB with NaN3. C, C. albicans treated with F-Hst5 in PPB with NaN3, subsequently washed in PPB with NaN3 to remove unbound Hst5 and then washed and resuspended in PPB with NaCl to wash-out NaN3. Controls incubated with F-CystS peptide were completely negative. Right column, adenine nucleotide content of C. albicans cells treated the same way, in the absence of Hst5.

 
The Cell Wall Is Not Involved in the Desensitizing Effects of Energy Depletion—To identify the site at which F-Hst5 accumulated in azide-treated C. albicans cells, we first attempted to study its localization by immunogold electron microscopy. In the presence of sodium azide, however, more than 90% of the cells were unlabeled, and in the remaining cells only a few gold particles were found with a seemingly preferred labeling of the cell wall (not shown). This scarce labeling, however, did not allow us to draw firm conclusions about the ultrastructural localization of F-Hst5. Therefore, we examined the association between C. albicans and the more potent Dhvar5, which is moderately sensitive to azide (Table 1). Confocal fluorescence microscopy revealed that in the absence of azide F-Dhvar5 labeled the interior of the cells (Fig. 5A). Some cells exhibited a granular staining pattern plus some surface labeling, whereas in other cells the label was distributed more uniformly and intensely throughout the interior. In the presence of azide, a shift in localization of F-Dhvar5 occurred, with now ~70% of the cells exhibiting also labeling at their boundaries (Fig. 5B). These cells were faintly intracellularly labeled with strong fluorescent spots observed in about 50% of the cells. The remaining 30% of the cells showed cytoplasmic labeling, similar to that observed in the absence of azide. Immunogold electron microscopy of F-Dhvar5-treated C. albicans (Fig. 5C) confirmed the predominant intracellular labeling patterns found by confocal microscopy. In addition, gold particles were occasionally found at the cell wall, but not in the plasma membrane. In the presence of sodium azide, however, the number of gold particles associated with the cell wall was substantially increased (Fig. 5D). Taken together these experiments revealed that in the presence of azide, the interaction of peptides with the cell was blocked at the cell wall. This was reminiscent to a previous report that in Saccharomyces cerevisiae stress proteins, expressed at the cell wall, conveyed resistance to osmotin, an antimicrobial peptide from the tobacco plant (24). This prompted us to examine whether removal of the cell wall would abolish the effect of azide. This, however, appeared not to be the case; spheroplasts were as sensitive to azide as whole cells, both in the PI assay (Fig. 6) and in the viability assay (not shown), excluding a role for the cell wall in conferring resistance to Hst5.


Figure 5
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  		FIGURE 5.
Localization of F-dhar5 in C. albicans. Suspensions of C. albicans (3.2 x 106 cells/ml) were preincubated at 37 °C for 15 min in PPB with NaCl or PPB with NaN3. F-Dhvar5 (17 µM) was added, and incubation was continued for 15 min. A, C. albicans treated with F-Dhvar5 in PPB with NaCl. B, C. albicans treated with F-Dhvar5 in PPB with NaN3. Controls incubated with F-CystS peptide were completely negative. C, immunoelectron microscopy of cells treated with F-Dhvar5 in PPB with NaCl. Cells treated with F-Dhvar5 in PPB with NaCl exhibit predominantly an intracellular labeling pattern (arrows). D, immunoelectron microscopy of cells treated with F-Dhvar5 in PPB with NaN3, showing labeling of the cell wall by Dhvar5. w, cell wall; m, cell membrane.

 


Figure 6
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  		FIGURE 6.
Effect of azide on Histatin 5-mediated PI influx in C. albicans spheroplasts. Suspensions of whole cells (C) or spheroplasts (S) of C. albicans (2 x 107 cells/ml) were incubated with PPB supplemented with 0.5 M sorbitol as osmoprotectant and NaCl or NaN3. After 30 min PI was added, and the cells were transferred to wells containing equal volumes of serially diluted Hst5 in the corresponding buffer. {diamondsuit}, spheroplasts in PPB with NaCl; {blacktriangleup}, whole cells in PPB with NaCl; *, spheroplasts in PPB with NaN3; {blacksquare}, whole cells in PPB with NaN3. Development of fluorescence was followed for each concentration of the peptides at 5-min intervals for 1 h at {lambda}ex 544 nm and {lambda}em 620 nm. The figure shows the fluorescence, expressed in arbitrary units (AU), after 1 h of incubation. The curves are representative of three independent experiments, carried out in duplicate.

 
The Plasma Membrane Potential Does Not Play a Role in the Azide-induced Decreased Sensitivity of C. albicans to Hst5—It has been reported that an inside negative membrane potential can enhance the activity of membrane-directed cationic antimicrobial peptides against living bacteria cells and against model membrane vesicles (25, 26). To establish whether reduction of the membrane potential plays a role in the azide-induced decreased sensitivity, we examined the effect of energy depletion on the membrane potential of C. albicans, using the potential-dependent distributional fluorescent dye DiSC3(5) (Fig. 7). Upon addition of DiSC3(5) to a C. albicans suspension, a fast decrease in DiSC3(5) fluorescence occurred, reflecting the potential-driven uptake of the probe by the yeast cells, accompanied by self-quenching of the fluorescence. Subsequent addition of Hst5 led to a steady rise in fluorescence, highlighting the dissipation of the membrane potential and concomitant leaking of the probe (Fig. 7A). The addition of azide to C. albicans cells preloaded with DiSC3(5) led to a small increase in DiSC3(5) fluorescence (Fig. 7B), likely because of the dissipation of only the mitochondrial membrane potential (3). This is in line with a previous study revealing that azide has little if any effect on the membrane potential of yeast cells (28). Subsequent addition of excess Hst5 to these azide-pretreated Candida cells did not result in further dissipation of the membrane potential (Fig. 7B). In contrast, the strongly membrane-disruptive peptide Dhvar4 (6, 27) induced a rapid dissipation of the membrane potential, both in the control cells and in the azide-pretreated cells (Fig. 7, C and D). These findings were in line with the results of Table 1, which showed similar differences between Hst5 and Dhvar4 with regard to their fungicidal activity to azide-treated C. albicans. Altogether, these experiments demonstrated that in the presence of azide the membrane potential was maintained for the larger part.

Energy Poisons Affect the Fluidity of the Membrane Probe DPH—Because of the intrinsic membrane-active features of cationic antimicrobial peptides, we next focused on the role of the membrane of the yeast cell and investigated whether energy depletion affected the physical properties, in particular the fluidity, of the phospholipid bilayer. To address this point, we treated C. albicans cells with the fluorescent membrane probe DPH, which is used to monitor the physical environment in different regions of the lipid bilayer. DPH is a rod-like molecule and partitions into the interior (hydrophobic core) of the bilayer. This partitioning is accompanied by an increase in fluorescence, because the quantum yield of DPH in the membrane is much higher than that in water (29). It was observed that upon the addition of azide or valinomycin to DPH-pretreated C. albicans, an immediate rise in DPH fluorescence occurred (not shown), indicating a change in the direct molecular environment of DPH. However, a number of conditions, including the fluidity, the water content, and the dielectric constant in the environment of the probe, influence the fluorescence properties of DPH, and this hampers an unambiguous interpretation of fluorescence intensity data. We therefore determined the effect of azide treatment on the steady state anisotropy of DPH. This parameter is inversely related to the membrane fluidity (30). For comparison, we tested the effect of agents known to influence the membrane fluidity, including the membrane-fluidizer benzyl alcohol (31) and the membrane-rigidifier Me2SO (Fig. 8). It was found that azide treatment induced an increase in the DPH anisotropy. Treatment with Me2SO induced an increase in the DPH anisotropy, in line with the anticipated rigidifying effect on the membrane. On the other hand benzyl alcohol caused a decrease in DPH anisotropy, reflecting an increase in membrane fluidity. Furthermore, benzyl alcohol reversed the effect of azide on the DPH anisotropy (Fig. 8). Hst5 treatment had no effect on the anisotropy value of DPH (not shown).

The Membrane Fluidizer Benzyl Alcohol Abolishes the Azide-induced Resistance to Hst5—We hypothesized that the membrane rigidifying effect of azide was the underlying cause of the resistance against Hst5 and therefore tested whether the fluidizer benzyl alcohol might reverse the protective effect of azide (Fig. 9). These experiments revealed that 75 mM benzyl alcohol completely restored the sensitivity of azide-treated C. albicans cells to Hst5, both in the PI assay (Fig. 9A) and in the viability assay (not shown). Furthermore, benzyl alcohol restored the cytoplasmic localization of Hst5 in azide-treated C. albicans cells (not shown). Benzyl alcohol did not restore the energy charge of the yeast cell, ruling this out as an explanation for the increased sensitivity (Fig. 9D). The inhibitory effects of high ionic strength, however, could not be abolished by benzyl alcohol, even at a 100 mM concentration (not shown). This indicates that the effect of azide occurs downstream from the initial, ionic strength-sensitive interaction between the positively charged Hst5 and the negatively charged surface of the yeast cell. At low temperature (4 °C) the activity of Hst5 was substantially diminished (Fig. 9B), whereas the energy content of the cells remained unchanged. This inhibition was reversed by 25 mM benzyl alcohol, underlining that the decreased fluidity of the membrane was responsible for the increased resistance against Hst5 at low temperature (Fig. 9B). The membrane rigidifying agent Me2SO increased the resistance to Hst5 at 30 °C, thus mimicking the effect of low temperature. In PPB supplemented with 10% (v/v) Me2SO, the Hst5 induced PI influx was inhibited for ~70% (Fig. 9C). Also in the viability assay was confirmed that Me2SO and low temperature protected against the fungicidal activity of Hst5 (not shown). Despite the relatively high Me2SO concentration, in the absence of peptides no enhancement in PI fluorescence occurred (Fig. 9C) nor a decrease in the energy charge (Fig. 9D), indicating that the integrity of the yeast plasma membrane was maintained. Culturing confirmed that treatment for 60 min with 10% Me2SO had no adverse effects on the viability of the yeast cells (not shown) in line with other reports (32).


Figure 7
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  		FIGURE 7.
Effect of azide on the plasma membrane potential of C. albicans. To a suspension of C. albicans (3.2 x 106 cells/ml) in PPB, DiSC3(5) was added to a final concentration of 1.5 µM. A, after a constant fluorescence level had been achieved (about 10 min), excess Hst5 was added, resulting in a steady dissipation of the membrane potential. B, addition of 5 mM NaN3 resulted in a small increase in fluorescence. Subsequent addition of excess Hst5 did not depolarize the membrane further. C, addition of excess Dhvar 4 resulted in a virtually complete depolarization of the membrane in the control cells. D, addition of Dhvar4 induced a virtual complete depolarization of the membrane in the presence of azide.

 


Figure 8
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  		FIGURE 8.
Change in DPH fluorescence anisotropy after treatment of C. albicans with azide, benzyl alcohol, and Me2SO. C. albicans were incubated with the membrane probe DPH for 20 min. The cells were washed in PPB to remove unbound probe, and suspended in PPB alone (control) and in PPB supplemented with 5 mM NaN3 (azide), 5 mM NaN3 with 50 mM benzyl alcohol (azide+BA), 50 mM benzyl alcohol (BA), and 5% Me2SO (DMSO). Relative changes in anisotropy value relative to PPB alone are plotted. *, p < 0.01 (relative to control). An increase in anisotropy reflects a decrease in membrane fluidity.

 
The Cytoskeleton Inhibitor Jasplakinolide Inhibits the Membranolytic Action of Hst5—The previous experiments revealed that low energy conditions induced changes in the cell membrane, rendering it less sensitive to membrane-active peptides such as Hst5. The molecular events, however, linking the energy charge of the cell with the membrane fluidity remain unclear. In a first attempt to identify the structures involved, we focused on major energy consuming systems in the cell. Because of the very short time scale of the effects (<1 min; Fig. 1), we deemed the involvement of transcriptional or translational processes unlikely and focused on major energy-consuming systems in the cell. One major energy drain in eukaryotic cells is the maintenance of the actin cytoskeleton, which in nerve cells and platelets is responsible for about 50% of the energy consumption (33). The turnover of the cytoskeleton is a continuously ongoing, energy-demanding process, which renders it directly sensitive to changes in the energy charge of the cell. Moreover, the cytoskeleton is connected at multiple points with the cell membrane, making direct communication with the membrane physically possible. We investigated therefore the effect of two membrane-permeating marine natural products, latrunculin B and jasplakinolide, which influence the turnover of the actin cytoskeleton (Fig 10). Latrunculin B sequesters actin monomers as they are released from filaments and prevents their reassembly, inducing depolymerization of the actin cytoskeleton. In contrast, jasplakinolide, by inhibiting the ATP-driven release of actin monomers, induces actin polymerization (34), thus mimicking energy depletion (35). At 100 µM concentration, latrunculin B had no effect on the Hst5-C. albicans interaction, neither in the PI assay (Fig 10A) nor in the viability assay (not shown). On the other hand, jasplakinolide inhibited in a dose-dependent way the Hst5-mediated influx of propidium iodide (Fig 10B). At 10 µM jasplakinolide, a 50% reduction in PI influx, which is a measure for membrane disintegration, was observed. By measuring the adenine nucleotide content of the C. albicans cells, it was verified that jasplakinolide did not decrease the ATP content of the cell (not shown), excluding that the observed inhibition was indirectly caused by energy depletion.


Figure 9
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  		FIGURE 9.
Effect of membrane-modulating agents and conditions on the sensitivity of C. albicans to Histatin 5. A, C. albicans cells were preincubated either in PPB with NaN3 or in PPB with NaCl. After 30 min, benzyl alcohol (BA) was added. After 15 min 2-fold serially diluted Hst5 was added, and PI influx was determined. Development of fluorescence was followed for each concentration of the peptide at 5-min intervals for 1 h at {lambda}ex 544 nm and {lambda}em 620 nm. The figure shows the fluorescence, expressed as arbitrary units, after 1 h. {blacksquare}, PPB with NaN3; {diamondsuit}, PPB with 5 mM NaN3 with 100 mM benzyl alcohol; {blacktriangleup}, PPB with 5 mM NaCl; {blacksquare}, PPB with 5 mM NaCl with 100 mM benzyl alcohol. B, C. albicans cells were incubated with serial dilutions of Hst5 in PPB supplemented with various concentrations of benzyl alcohol, at 4 °C (no azide). PI was added, and after 1 h the fluorescence was measured at {lambda}ex 544 nm and {lambda}em 620 nm. *, PPB; •, PPB with 12.5 mM benzyl alcohol; {blacktriangleup}, PPB with 25 mM benzyl alcohol; {diamondsuit}, PPB with 50 mM benzyl alcohol. C, C. albicans cells were suspended in PPB supplemented with varying Me2SO (DMSO) concentrations (no azide). PI was added and cells were transferred to wells containing equal volumes of serially diluted Hst5 dissolved in the corresponding buffers. The figure shows the fluorescence, expressed as arbitrary units, after 1 h. {blacksquare}, PPB; {blacktriangleup}, PPB with 1% Me2SO; {diamondsuit}, PPB with 2.5% Me2SO; *, PPB with 5% Me2SO; •, PPB with 10% Me2SO. The curves are representative of two independent experiments, carried out in duplicate. D, C. albicans was incubated in PPB at 4 and 30 °C for 30 min. In addition, C. albicans was incubated in PPB supplemented with 10% Me2SO, 5 mM NaN3, and 5 mM NaN3 with 100 mM benzyl alcohol at 30 °C. After 30 min, the cells were lysed by transfer into boiling ethanol. Adenine nucleotide content was determined by CZE. Black bars, ATP; shaded bars, ADP; white bars, AMP. No measurable amounts of adenine nucleotide were released in the supernatant of intact untreated cells in the time course of these experiments. The data are representative of at least two independent experiments, carried out in duplicate. The values represent the means of duplicate measurements.

 


Figure 10
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  		FIGURE 10.
Effect of the cytoskeleton inhibitors latrunculin B and jasplakinolide on the sensitivity of C. albicans for Histatin 5. A, C. albicans were preincubated for 30 min in PPB supplemented with 100 µM concentrations of latrunculin B (latB). PI was added, and the cells were transferred to wells containing equal volumes of serially diluted Hst5 in PPB supplemented with latrunculin B in the corresponding concentration. Development of fluorescence was monitored for each concentration of the peptides at 5-min intervals for 1 h at {lambda}ex 544 nm and {lambda}em 620 nm. The figure shows the fluorescence, expressed in arbitrary units, after 1 h of incubation. The data are representative of three independent experiments, carried out in duplicate. B, C. albicans were preincubated for 30 min in PPB supplemented with various concentrations of jasplakinolide (jasplak). PI was added, and the cells were transferred to wells containing equal volumes of serially diluted Hst5 in PPB, supplemented with jasplakinolide in the corresponding concentration. The development of fluorescence was followed for each concentration of the peptides at 5-min intervals for 1 h at {lambda}ex 544 nm and {lambda}em 620 nm. The figure shows the fluorescence, expressed in arbitrary units, after 1 h of incubation. The data are representative of three independent experiments, carried out in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Lowering the energy content of C. albicans affects the activity of a wide variety of peptides (Table 1), indicating that this effect is not restricted to the Hst5-C. albicans interaction. Similarly, various processes taking place at the membrane of eukaryotic cells, including the entry of polyarginine and polylysine into fibroblasts (36, 37), the endocytosis of TAT peptides into HeLa cells (38), and influenza virus budding from the plasma membrane of infected kidney cells (39), are inhibited by energy depletion. On the other hand, the sensitivity of Escherichia coli to Hst5 is not changed by azide,3 despite the fact that this bacterium, like C. albicans, does contain an azide-sensitive respiratory chain. The common intuitive explanation of how energy poisons decrease the activity of these peptides is that peptide entry into the cell requires the presence of active transport systems or a membrane potential. This implies that the energy charge of the cell, directly or indirectly, drives translocation of peptides across the cell membrane with the concomitant toxic consequences. Support for the involvement of the membrane potential has come from model studies showing that the induction of a membrane potential led to enhanced peptide translocation into protein-free lipid vesicles (26, 40). However, in agreement with other studies (28), we found that blocking the mitochondrial respiration did not diminish the plasma membrane potential of C. albicans cells, whereas the entry of Hst5 into the cell was still abolished. In line, the potassium ionophore valinomycin and the protonophore CCCP, neither of which affect the plasma membrane potential in yeast (41–43), almost completely abolished the sensitivity to Hst5. In this study we found that energy depletion affected the physical state of the plasma membrane, as reflected by an increased value of the DPH anisotropy. Because increasing the fluidity by benzyl alcohol reversed both the effect of azide on the Hst5-mediated membrane disruption and on the membrane fluidity (Figs. 8 and 9A), it is tempting to conclude that not energy depletion itself but rather its effect on the physical state of the membrane causes the protective effect of energy poisons. Rigidifying the membrane by Me2SO or low temperature likewise decreased the membranolytic activity of Hst5, albeit to a smaller extent. Membrane rigidifying effects of energy depletion have previously been reported by Haidekker et al. (44) using a laser-based scatter system and by Florine-Casteel et al. (45) using video fluorescence polarization microscopy. The latter authors reported that the plasma membrane of cultured rat hepatocytes becomes uniformly rigid within a few minutes after the addition of metabolic inhibitors, when ATP levels have fallen by >95%. These authors speculated that increased phospholipase activity leading to an altered cholesterol/phospholipid ratio or lipid peroxidation might account for the increased lipid order. However, in our opinion the fast kinetics (<5 min; Fig. 1) combined with the ready reversibility of these effects (Figs. 2 and 4), make it less likely that an increase in activity of phospholipid-modifying enzymes would be the primary mechanism underlying the observed phenomena.

Although the molecular principles linking the energy status with the membrane properties of the cell and its sensitivity to antimicrobial peptides are not completely understood, a number of observations point to a role for the actin cytoskeleton. The cytoskeleton inhibitor jasplakinolide, which promotes actin polymerization, concurrently renders Candida less sensitive to membrane disruption by Hst5, thus having the same overall effect as energy poisons but without lowering the ATP levels. Maintenance of the cytoskeleton is, also in yeast (46), an ongoing process that requires a continuous and substantial supply of ATP. In human platelets and nerve cells this may require as much as 50% of the energy sources of the cell (33). This metastable situation is inherently sensitive to energy depletion, as illustrated by the observation that decreased intracellular ATP levels are associated with an immediate increase in the fractions of polymerized actin observed in vivo and in vitro (34), an effect that partly can be mimicked by jasplakinolide. In line with the present findings, Sheikh et al. (47) found that jasplakinolide caused a marked increase in cell rigidity in neutrophils. Because of the abundant connections between the actin cytoskeleton and the plasma membrane, cytoskeletal rearrangements can induce direct changes in cell and membrane morphology (48–51). In Dictyostelium cells, for example, depletion of cellular ATP by azide causes a "rigor" contraction of the cytoskeleton, which renders the cells stiffened and spherical in appearance (52). Such cytoskeletal rearrangements occur in minutes (53), a time scale on which energy poisons depleted the energy charge of the cell (Fig. 2A), induced resistance against Hst5 (Fig. 1B), and affected the physical state of the membrane.

In the present study we investigated the molecular mechanism underlying the long known phenomenon that energy depletion in yeast induces resistance against cationic peptides and proteins (1). At variance with the generally accepted explanation that implies a role for ATP-driven transport systems, we found that energy depletion induces membrane rigidification, possibly mediated by the cytoskeleton, which desensitizes C. albicans to antimicrobial peptides. Because of the similar responses of yeast and mammalian cells toward energy depletion, the peptide-yeast model may provide a new model system to study the early cellular events triggered by ATP depletion, e.g. in hypoxia, or in ischemic attacks.


   FOOTNOTES
 
* 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

1 Member of the Skeletal Tissue Engineering Group Amsterdam. To whom correspondence should be addressed: Academic Centre for Dentistry Amsterdam, Dept. of Oral Biochemistry, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel.: 31-20-4448672; Fax: 31-20-4448685; E-mail: eci.veerman{at}vumc.nl.

2 The abbreviations used are: Hst5, Histatin 5; FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PPB, potassium phosphate buffer; PI, propidium iodide; FACS, fluorescence-activated cell sorter; DPH, diphenylhexatriene. Back

3 E. C. I. Veerman, unpublished observations. Back


   ACKNOWLEDGMENTS
 
The drosocin peptides were a kind gift from Dr. F. J. Bikker (TNO, Rijswijk, The Netherlands).



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 DISCUSSION
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