Energy Depletion Protects Candida albicans against Antimicrobial Peptides by Rigidifying Its Cell Membrane

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. 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. 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 (1Olson V.L. Hansing R.L. McClary D.O. Can. J. Microbiol. 1977; 23: 166-174Crossref PubMed Scopus (12) Google Scholar, 2Lehrer R.I. Ganz T. Szklarek D. Selsted M.E. J. Clin. 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Chabasso D. Bouchara J-P. Antimicrob. Agents Chemother. 2003; 47: 847-853Crossref PubMed Scopus (53) Google Scholar). In Chlorella metabolic inhibition abolishes the membrane-disruptive effects of the polyene nystatin and even of the detergent Triton X-100 (8Komor E. Weber H. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1814-1818Crossref PubMed Scopus (33) Google Scholar). 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 (1Olson V.L. Hansing R.L. McClary D.O. Can. J. Microbiol. 1977; 23: 166-174Crossref PubMed Scopus (12) Google Scholar, 9Aspedon A. Groisman E.A. Microbiology. 1996; 142: 3389-3397Crossref PubMed Scopus (66) Google Scholar, 10Lehrer R.I. Barton A. Daher K.A. Harwig S.S.L. Ganz T. Selsted E. J. Clin. Investig. 1989; 84: 553-561Crossref PubMed Scopus (609) Google Scholar, 11Falla T.J. Karunaratne D.N. Hancock R.E.W. J. Biol. Chem. 1996; 271: 19298-19303Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 12Kordel M. Benze R. Sahl H.G. J. Bacteriol. 1988; 170: 84-88Crossref PubMed Google Scholar). 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 (13White T.C. Marr K.A. Bowden R.A. Clin. Microbiol. Rev. 1998; 11: 382-402Crossref PubMed Google Scholar). 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) 2The 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. -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. 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-α-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 (3Helmerhorst E.J. Breeuwer P. Van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). 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.TABLE 1The effect of energy depletion on the activity of various antimicrobial peptidesPeptideSequenceLC50NaClNaN3μmHistatin 5DSHAKRHHGYKRKFHEKHHSHRGY1.1>100F-Histatin 53.0>100Dh5 (11-24)KRKFHEKHHSHRGY2.5>100P-113 (4-15)AKRHHGYKRKFH2.7>100P-113Dakrhhgykrkfh2.4>100Dhvar4KRLFKKLLKFSLRKY0.10.2Dhvar5LLLFLLKKRKKRKY210F-Dhvar52.56.5LFampin 265-284DLIWKLLSKAQEKFGKNKSR0.82.0LFcin 17-30FKCRRWQWRMKKLG0.5>40Drosocin-1GKPRPYSPRPTSHPRPIRV3.5>100Drosocin-4GKPRPYTPRPTSHPRPIRV2.3>100 Open table in a new tab 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 β-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 (14Veerman E.C.I. Nazmi K. Van't Hof W. Bolscher J.G.M. Den Hertog A.L. Nieuw Amerongen A.V. Biochem. J. 2004; 381: 447-452Crossref PubMed Scopus (67) Google Scholar). 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 (3Helmerhorst E.J. Breeuwer P. Van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). 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 (15Eddy A.A. Hopkins P.G. Biochem. J. 1985; 231: 291-297Crossref PubMed Scopus (27) Google Scholar). 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 (3Helmerhorst E.J. Breeuwer P. Van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). 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 × g), and resuspended in the same buffer. The resulting suspensions were divided into two equal volumes, which were centrifuged (5 min at 10,000 × 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 100× NA 1.4 object lens. Electron Microscopy—Ultrastructural localization was examined using immunogold-labeling and transmission electron microscopy as described previously (6Ruissen A.L.A. Groenink J. Van't Hof W. Walgreen-Weterings E. Van Marle J. Van Veen H. Voorhout W.F. Veerman E.C.I. Nieuw Amerongen A.V. Peptides. 2002; 23: 1391-1399Crossref PubMed Scopus (26) Google Scholar). 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 (6Ruissen A.L.A. Groenink J. Van't Hof W. Walgreen-Weterings E. Van Marle J. Van Veen H. Voorhout W.F. Veerman E.C.I. Nieuw Amerongen A.V. Peptides. 2002; 23: 1391-1399Crossref PubMed Scopus (26) Google Scholar). 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 (14Veerman E.C.I. Nazmi K. Van't Hof W. Bolscher J.G.M. Den Hertog A.L. Nieuw Amerongen A.V. Biochem. J. 2004; 381: 447-452Crossref PubMed Scopus (67) Google Scholar). 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) (16Gonzales B. François J. Renaud B. Yeast. 1997; 13: 1347-1356Crossref PubMed Scopus (297) Google Scholar) 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 × 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 (17Obrénovitch A. Sené C. Nègre M-T. Monsigny M. FEBS Lett. 1978; 88: 187-191Crossref PubMed Scopus (21) Google Scholar, 18Kuhry J.G. Duportail G. Bronner C. Laustriat G. Biochim. Biophys. Acta. 1985; 845: 60-67Crossref PubMed Scopus (186) Google Scholar). 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 (19Patra D. J. Appl. Spectrosc. 2004; 71: 334-338Crossref Scopus (4) Google Scholar). 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. Effect of Energy Metabolism on Candidacidal Activities of Antimicrobial Peptides—In previous studies we found that Hst5 accumulates in the mitochondria of C. albicans (3Helmerhorst E.J. Breeuwer P. Van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 20Ruissen A.L.A. Groenink J. Helmerhorst E.J. Walgreen-Weterings E. Van't Hof W. Veerman E.C.I. Nieuw Amerongen A.V. Biochem. J. 2001; 356: 361-368Crossref PubMed Scopus (86) Google Scholar). Because treatment with mitochondrial poisons such as sodium azide renders C. albicans cells insensitive to Hst5 (3Helmerhorst E.J. Breeuwer P. Van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 20Ruissen A.L.A. Groenink J. Helmerhorst E.J. Walgreen-Weterings E. Van't Hof W. Veerman E.C.I. Nieuw Amerongen A.V. Biochem. J. 2001; 356: 361-368Crossref PubMed Scopus (86) Google Scholar), 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 (21Van der Kraan M.I.A. Van Marle J. Nazmi K. Groenink J. Van 't Hof W. Veerman E.C.I. Bolscher J.G.M. Nieuw Amerongen A.V. Peptides. 2006; 26: 1537-1542Crossref Scopus (68) Google Scholar) LFcin 17–30 and LFampin 265–284; (iii) drosocin and a drosocin variant (22Bikker F.J. Kaman-van Zanten W.E. De Vries-Van de Ruit A.-M.B.C. Voskamp-Visser I. Van Hooft P.A.V. Mars-Groenendijk R.H. De Visser P.C. Noort D. Chem. Biol. Drug Des. 2006; 68: 148-153Crossref PubMed Scopus (40) Google Scholar); 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 (14Veerman E.C.I. Nazmi K. Van't Hof W. Bolscher J.G.M. Den Hertog A.L. Nieuw Amerongen A.V. Biochem. J. 2004; 381: 447-452Crossref PubMed Scopus (67) Google Scholar). 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).FIGURE 2The effect of energy poisons on the energy charge of C. albicans. A, C. albicans (2 × 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). ♦, ATP; ▴, 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 × 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. ♦, ATP; ▴, 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (23Li X.W.S. Sun J.N.N. Okamoto-Shibayama K. Edgerton M. J. Biol. Chem. 2006; 281: 22453-22463Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 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