INTRODUCTION

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Oral candidiasis is a frequent superficial infection in humans associated with mechanical or traumatic factors or with an immunocompromised host (1). Candida albicans is the predominant species of yeast isolated from oral tissues of patients with oral candidiasis (2). Oral candidiasis is commonly associated with HIV¹ infection and is a criterion for the development and progression of AIDS (3). Relatively few antifungal drugs are available for clinical treatment of oral or systemic candidiasis. Increased use of these antifungal agents to treat candidiasis in late stage AIDS and cancer patients has resulted in a dramatic increase in emergence of candidal species with antifungal drug resistance, especially to azole-based drugs.

Growth or adhesion of pathogenic yeasts as well as bacteria in the oral cavity are regulated by glandular secretion of an array of salivary proteins (4, 5). Histatins (Hsts) are structurally related basic proteins of acinar cell origin, which possess in vitro candidacidal and candidastatic (6-8) activities and, to a lesser degree, bactericidal properties (9) and participate in nonimmune host defense of the oral cavity (10). Hsts have potential as therapeutic agents against oral candidiasis, being potent antifungal molecules with no toxicity to humans.

Histatin 1 (Hst 1), histatin 3 (Hst 3), and histatin 5 (Hst 5) are the major Hsts products found in saliva from human submandibular-sublingual and parotid glands (11, 12) and are present in healthy adults at concentrations of 50-425 µg/ml (13). Hst 1 and Hst 3 are the full-length precursor molecules, which are cleaved by specific proteolytic processing events during secretion to produce smaller histatins (14-16). In vitro, Hst 5 is the most potent candidacidal member of the family that kills pathogenic Candida species from 90% to 100% at physiological concentrations (15-30 µM) (10, 17). We have shown that Hst M, the middle portion of Hst 3 (residues 9-24), has equivalent candidacidal activity with the full-length molecule (17). Candidacidal activity of this fragment has been confirmed (8), and duplication of these residues (residues 13-24) as a tandem repeat within Hst 3 has produced a peptide with enhanced candidacidal activity at low concentrations (18).

Histatins should be added to the described families of natural antibiotics produced by host tissues in contact with indigenous microorganisms (19). These antimicrobial proteins are produced by a variety of tissues and organisms as a means of nonimmune host defense. Although Hsts have similar size and net positive charge as other naturally occurring antimicrobial peptides, they possess structural features unique from other described low molecular weight cationic proteins. The high histidine content, lack of disulfide bonds, and weak amphipathic character of the -helical structures distinguish Hsts from the known natural antimicrobial proteins such as defensins, bactenecins, and cecropins (19).

The mode of action of a number of cationic host defense polypeptides is related to increased membrane permeability and disruption of microbial cell structure. Studies with artificial lipid bilayers have demonstrated a range of effects including channel formation by defensins (20-22) and disruption of lipid packing by bactenecins (23), dermaseptin (24), nisin (25), cecropin (26), and tachyplesin (27), which results in permeabilization of the membrane. Lytic properties have also been described for the polyene antimycotics, which are currently used drugs in treatment of candidiasis. Polyene antimycotics form pores in the plasma membrane by complex formation with ergosterol of the plasma membrane (28, 29), resulting in loss of potassium and leakage of other cell products. These drugs have additional secondary effects on inhibition of plasma membrane ATPase leading to proton efflux as well as reduction of activity of many biosynthetic enzymes (29). The synthetic azole derivatives are the other major class of antifungal drugs. The azole-based drugs act on yeast mitochondria and through a complex multistep pathway inhibit the biosynthesis of ergosterol, the major sterol of yeast plasma membranes (30, 31).

The mechanisms by which salivary Hsts exert candidacidal activity are not well understood. Electron micrographs of C. albicans cells following Hst treatment revealed damage at the cell membrane, as well as the presence of cytoplasmic vacuoles, suggesting loss of intracellular materials (32). In addition, C. albicans cells were found to release potassium in the presence of a fraction of histidine-rich polypeptides purified from human parotid saliva, indicating a loss of yeast membrane integrity (33). Further, Hsts were effective in killing azole-resistant Candida species, thus implicating a different mechanism of Hst action from that of the azole-based antifungal drugs (34, 35). Studies using Hst 5 variants with one or two amino acid substitutions showed that His¹⁸, Lys¹³, Arg²², and the dipeptide sequence Phe¹⁴/His¹⁵ may be important for optimal activity (36). Moreover, Hst 5 variants with reduced killing ability exhibited similar helical content to Hst 5, suggesting that the -helical conformation alone is not solely responsible for optimal candidacidal activity (36).

Although earlier potassium release results suggested that Hsts' biological effects may be related to direct peptide-membrane interactions, recent structural studies of the active fragment of Hst (37) and two-dimensional NMR studies of Hst 5 (38) revealed that the weak amphipathic character of the helical structure precludes spontaneous insertion into the membrane. The size of the active fragment (16 residues) also argues against its ability to function as a channel-forming peptide. Moreover, the yeast cell wall is rich in mannoproteins and glucans containing anionic sites, which may form electrostatic complexes with Hsts and prevent interaction with the cell membrane.

Hsts' potent antifungal activity, lack of toxicity to humans, and ability to kill azole-resistant yeast strains underscore the importance of detailed understanding of their mechanism of action. In this study, we examined whether Hsts can induce direct physicochemical damage to yeast cell membranes (cationic peptide model) or whether binding to a specific yeast component is required for Hst killing by performing biological and fluorescent dye release assays, Hst binding, and cross-linking studies on C. albicans intact cells and spheroplasts.

	EXPERIMENTAL PROCEDURES

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Materials-- C. albicans strain DS1 was isolated from the palate of a denture stomatitis patient (39) and Saccharomyces cerevisiae strains TI#20 and BJ2169 were obtained from Dr. J. Bruenn (Department of Biological Sciences, SUNY, Buffalo, NY). Sabouraud dextrose agar and YPD media were from Difco. Wang resins and Fmoc amino acid derivatives were purchased from Calbiochem (NovaBiochem Co., La Jolla, CA) and Sigma. Na¹²⁵I was purchased from Amersham Pharmacia Biotech. Zymolyase-20T was from ICN Biomedicals (Costa Mesa, CA). Calcein-AM and calcein were obtained from Molecular Probes (Eugene, OR). Egg yolk phosphatidylcholine (ePC) and phosphatidylserine (PS) were obtained from Avanti Polar Lipids (Birmingham, AL); cholesterol and ergosterol were purchased from Sigma. Sephadex G-25 and Sephadex G-10 were from Amersham Pharmacia Biotech; disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), and BCA protein reagent were purchased from Pierce, and the electrophoretic reagents were from Bio-Rad.

Peptide Synthesis and Purification-- Full-length Hst 3 (DSHAKRHHGYKRKF HEKHHSHRGYRSNYLYDN) containing N-terminal (N = DSHAKRHH), middle active (M = GYKRKFHEKHHSHRGY), and C-terminal (C = RSNYLYDN) regions and Hst 5 (DSHAKRHHGYKRKFHEKHHSHRGY) containing N and M regions were synthesized using a Beckman System 990 synthesizer, standard solid phase synthesis protocols, and Fmoc chemistry (Table I). Peptides were cleaved from the resin and deprotected under anhydrous conditions using trifluoroacetic acid, 13% triethylsilane, and 6% phenol. Hst 4 (RKFHEKHHSHRGYRSNYLYDN) and Hst 4a (GYKRKFHEKHHSHRGYRSNYLYDN) containing M and C regions, region M (GYKRKFHEKH HSHRGY), and peptide region C (RSNYLYDN) were synthesized using t-butoxycarbonyl chemistry as described previously (17, 37).

Yeast Culture Conditions-- C. albicans was maintained on Sabouraud dextrose agar plates as described previously (17), and S. cerevisiae was maintained on YPD agar plates. C. albicans was inoculated into 10 ml of sucrose-salts-biotin yeast synthetic medium (SSB) (17, 41), and S. cerevisiae was inoculated into 10 ml of YPD medium. Cells were grown to stationary phase for 48 h at 25 °C with rotary agitation at 200 rpm. Blastoconidial cell morphology and cell numbers were determined by phase contrast microscopy using a cell counting chamber.

Conversion of Yeast Cells to Spheroplasts-- C. albicans cells were grown for 48 h at 25 °C in SSB medium and converted to spheroplasts with Zymolyase 20T (ICN Biomedicals, Inc.) as described (42). Briefly, C. albicans cells from 10-ml cultures were harvested by centrifugation at 600 × g, washed twice with TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA), and resuspended in 900 µl/g wet weight cells spheroplasting buffer (10 mM Tris, pH 7.4, 1 mM EDTA, and 1 M sorbitol as an osmotic protector). After the addition of 2-mercaptoethanol to a final concentration of 30 mM and 150 units Zymolyase-20T/g wet weight yeast cells, cells were incubated for 1-1.5 h at 30 °C with gently shaking. Spheroplast formation was monitored microscopically by lysis of osmotically sensitive cells in 5% SDS; under these conditions, more than 90% of the cells were converted to spheroplasts. Spheroplasts were washed twice and resuspended in buffer containing 1 M sorbitol for further use. In some cases, spheroplasts were solubilized in Laemmli sample buffer and subjected to SDS-PAGE.

Candidacidal Assay-- Antifungal activity of Hsts was examined by microdilution plate assay as described previously (17) with the following modifications. Fungicidal assays were performed on stationary C. albicans cells in the presence or absence of Hst 5, Hst 3, Hst 4, Hst M, Hst 4a, or Hst 3 C (0.98-125 µM) or 500 µM amylase, cytochrome c, and insulin--chain. Briefly, C. albicans cells were washed twice with 10 mM sodium phosphate buffer (Na₂HPO₄/NaH₂PO₄), pH 7.4, and resuspended at 1.8 × 10⁵ cells/ml. Cell suspensions (20 µl) were mixed with 20 µl of 10 mM phosphate buffer, pH 7.4, containing the indicated proteins and incubated for various times at 37 °C with shaking. Control cultures were incubated with 20 µl of 10 mM phosphate buffer alone. The reaction was stopped by addition of 360 µl of yeast nitrogen base (YNB); 40 µl of the suspension (360 cells) were spread onto Sabouraud dextrose agar plates and incubated for 48 h at 37 °C. In some experiments, candidacidal assays were performed in 10 mM phosphate buffer, pH 7.4, with 1 M sorbitol. In preliminary experiments, Hst 5 candidacidal activity was tested in 10 mM phosphate buffer containing either 1 M sorbitol, 2 mg/ml BSA, 0.6 M KCl, 0.5 M NaCl, or in 40 mM HEPES. Hst 5 activity was unaffected in 10 mM phosphate buffer containing 1 M sorbitol or 2 mg/ml BSA; however, it was completely inhibited in buffers containing high salt concentration or in HEPES buffer and therefore these reagents were not used throughout the experiments. Candidacidal assays were performed in duplicate or triplicate. Loss of viability was calculated as [1  (colonies from suspension with peptide/colonies from suspension with no protein)] × 100. The Hst concentration required to kill 50% of colony-forming unit yeast cells (LD₅₀) was calculated from the dose-response curves. Statistical significance of the results was calculated using Student's t test.

Cell Permeability Assay-- Cell permeability was monitored by release of the intracellular dye calcein. Following cellular uptake of calcein acetoxymethyl ester (calcein-AM), intracellular esterases convert the molecule into a nonpermeable acid form of calcein. Intracellularly, high concentration of dye leads to energy exchange between calcein molecules resulting in quenching. Leakage of calcein into the extracellular media was detected by measuring characteristic fluorescence emission spectra. Calcein-AM loading of C. albicans cells was performed on early stationary phase yeast cell cultures. Cells were washed twice with 10 mM phosphate buffer and resuspended at 10⁷cells/ml. C. albicans (10⁷cells) were loaded with 5 µl of 1 mM calcein-AM at a final concentration of 5 µM for 2 h at room temperature. Cells were washed four times to remove unincorporated dye, and 100 µl (10⁶) of cells were transferred to a quartz microcuvette filled with 300 µl of 10 mM phosphate buffer. In some experiments, whole cells were loaded with calcein-AM and subsequently converted to spheroplasts as described above and measurements were performed in 10 mM phosphate buffer containing 1 M sorbitol. Hst 5 (50 or 200 µM) was then added to calcein-loaded cells, and the fluorescence intensity of the induced calcein release (I_r) was recorded every 5 min at excitation and emission wavelengths of 485 and 530 nm, respectively, in a Hitachi F-2000 fluorescent spectrophotometer. Experiments were run for 60 or 90 min. The optimal number of cells, incubation time, and calcein-AM concentration were determined by preliminary experiments to reduce potential artifacts from overloading, dye leakage, and toxic effects. Fluorescence intensity corresponding to 100% of potentially available calcein (I_int) was determined by boiling the cells for 10 min. To confirm that all intracellular calcein was counted, calcein-loaded whole cells were converted to spheroplasts, lysed in boiling water, and total fluorescence measured. Total fluorescence released by lysis of osmotically sensitive cells was very similar to that released following boiling of whole cells. Total fluorescent intensity of the cell population following boiling was assumed to be equivalent to total potentially available intracellular calcein (I_int). The fluorescence intensity of calcein released from loaded cells before the addition of peptides was measured as background (I_bkg). Only cell preparations that showed less than 0.1% changes in the background over 10 min were used for further assays. Calcein efflux was calculated as percent fluorescent release = [(I_r  I_bkg)/(I_int  I_bkg)] × 100.

Preparation of Phospholipid Vesicles-- The reverse-phase evaporation method of vesicle formation (43, 44) was used to prepare small unilamellar vesicles using ePC or PS in liposome buffer (150 mM NaCl, 10 mM TES, pH 7.5) and 25 mM calcein. For liposomes containing sterols, cholesterol or ergosterol were recrystallized twice from ethanol and were incorporated into ePC and PS preparations at a lipid/sterol ratio of 2:1. Liposomes were extruded through 0.1-µm pore polycarbonate membranes using a high pressure extruder to produce uniformly sized small unilamellar vesicles. Liposomes were then separated from unencapsulated calcein by gel chromatography with a Sephadex G-25 column by eluting with liposome buffer. The phospholipid concentration of collected fractions containing liposomes was determined by phosphorus assay (45). The lipid concentration of each liposome preparation was 1-2 mM. Liposome preparations were adjusted to 0.1 mM phospholipid in liposome buffer, and background fluorescence monitored for 30 min to ensure stability of each preparation. Hst 5, Hst M, and Bac 5 were added to the liposome preparations at varying lipid to peptide concentration ratios and fluorescence of the released dye was recorded as described above. Liposomes were then lysed with 0.5% Triton X-100 to establish total fluorescent content of each preparation. Percent fluorescent release was calculated as [(I_r  I_bkg)/(I_int  I_bkg)] × 100.

Iodination of Histatin 5-- Synthetic histatin 5 (125 µg) was dissolved in 10 mM sodium phosphate buffer, pH 7.8, and radioiodinated using a chloramine T method as described previously (46). Free iodine was separated from ¹²⁵I-Hst 5 on Sephadex G-10 column that had been pre-equilibrated with 1 mg/ml BSA. The specific activity of the radioiodinated Hst 5 was typically around 20 cpm/fmol. The purity of radiolabeled Hst 5 was assessed by 15% SDS-PAGE using a Tris-Tricine electrode buffer and appeared as a single band. Based on the specific radioactivities of Na¹²⁵I (~15.5 mCi of ¹²⁵I/µg of iodine) and ¹²⁵I-Hst 5 (~20 cpm/fmol), the iodotyrosines were estimated to be less than 2% of the tyrosine residues in the final product. Nonradioactive iodination of Hst 5 was performed parallel to the radioactive labeling, except that 900 ng of Na¹²⁷I was used in place of Na¹²⁵I. Fractions collected after Sephadex G10 chromatography were concentrated by lyophilization and dissolved in 10 mM phosphate buffer, pH 7.4. Protein concentration was determined by amino acid analysis. ¹²⁷I-Hst 5 dissolved in either 10 mM phosphate buffer, pH 7.4, or 10 mM phosphate buffer with 2 mg/ml BSA was further tested for biological activity on candidacidal bioassay.

Binding Studies-- ¹²⁵I-Hst 5 binding was performed on C. albicans cells that were grown for 48 h in SSB medium. Cells were washed twice with 10 mM sodium phosphate buffer, pH 7.4, and resuspended at 4 × 10⁷ cells/ml in binding buffer (10 mM phosphate buffer, pH 7.4, 2 mg/ml BSA). Suspension assays were performed at cell densities of approximately 10⁶ cells (25 µl) for each point in a final volume of 50 µl binding buffer containing ¹²⁵I-Hst 5 for 30 min at room temperature. Following centrifugation (5000 × g, 3 min) and two rinses with binding buffer, the ligand-bound cell pellet was counted in a  counter at 55% efficiency. Specific binding was calculated as total minus nonspecific, where nonspecific binding (typically 15-28% of the total ligand bound) was determined in the presence of 100-1000-fold excess of unlabeled Hst 5. Competitive inhibition binding studies were carried out in binding buffer containing various concentrations (100 nM to 500 µM) of Hst 5, Hst 3, or Hst 4; plus 500 µM amounts of cytochrome c, insulin--chain, salivary amylase, or MUC7-derived peptides. Optimal cell number, incubation time, and temperature were determined by preliminary experiments. Cells were assayed for each experimental point in triplicate. The competition curves were fitted to the experimental points using the equation for one binding site using curve fit program LIGAND (Munson, NIH) and Kaleidagraph (Synergy System, Reading, PA).

Cell Fractionation-- C. albicans cells were grown for 48 h in SSB, harvested by centrifugation, and washed twice with 10 mM phosphate buffer, pH 7.4. Cells (0.7-1.0 g wet weight) were resuspended in 15 ml of cold homogenization buffer (10 mM phosphate buffer, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml benzamidine) and added to a 50-ml Bead Beater Chamber (Biospec, Bartlesville, OK) containing 15 ml w/v of prechilled 0.5-mm glass beads. Homogenization was carried out in five 1-min bursts at 4 °C. The beads were rinsed twice with 5 ml of homogenization buffer, and the lysate was centrifuged for 5 min at 1000 × g at 4 °C to remove unbroken cells and cell debris. Crude membrane fraction was separated from the soluble proteins by centrifugation for 1 h at 170,000 × g. The proteins from whole cell lysate (the low speed supernatant from the first centrifugation) were precipitated overnight at 20 °C with six volumes of ice-chilled acetone and centrifuged for 30 min at 14,000 rpm, and the dried pellets were solubilized in Laemmli sample buffer. The solubilized proteins from whole cell lysates or membrane fractions were separated on 7.5% or 10% SDS-PAGE and transferred to nitrocellulose membrane for overlay assay.

Overlay Assays-- Nitrocellulose membranes were blocked for 2 h in 10 mM Tris-HCl, pH 7.4, with 1% BSA and then incubated for 4 h with 50 nM ¹²⁵I-Hst 5 in binding buffer. Blots were washed with 10 mM Tris-HCl, pH 7.5, containing 0.1% Tween 20, and autoradiography was performed by exposing dried blots at 80 °C for 1-5 days on X-Omat film (Eastman Kodak Co.).

Cross-linking Studies-- Washed C. albicans cells were resuspended in binding buffer at 1.8 × 10⁹ cells/ml, and 150 µl (3 × 10⁸ cells) were incubated for 20 min at room temperature with 100 nM ¹²⁵I-Hst 5 in a final incubation volume of 200 µl. Where indicated, a 1000-fold excess of Hst 5 was added as an unlabeled competitor. Cells were then placed on ice and subsequently incubated for 20 min with 0.9 mM non-cleavable membrane-permeable DSS or membrane-impermeable BS cross-linking reagents (47), followed by three washes with binding buffer. Yeast protein extract was prepared from the ¹²⁵I-Hst 5 cross-linked cells by enzymatic digestion of the cell wall and subsequent lysis of the osmotically sensitive wall-free cells. Cross-linked C. albicans cells were resuspended in 200 µl of spheroplasting buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 M sorbitol, 480 µg of Zymolyase-20T, 30 mM 2-mercaptoethanol) and incubated for 1 h at 30 °C with gentle shaking. Wall-free cells were separated from the released cell wall material by centrifugation, solubilized in Laemmli sample buffer, boiled, and subjected to SDS-PAGE. Autoradiography was performed by exposing dried gels at 80 °C for 5-15 days.

	RESULTS

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Histatin-induced Loss of Yeast Cell Viability-- The antifungal activity of the major members of the Hst family has been characterized and active concentrations for candidacidal activity determined (6, 11, 17). We have confirmed and extended these results by testing the concentration dependence of synthetic Hsts representing various regions of the major Hsts (Table I) on C. albicans whole cells and spheroplasts and on the related yeast S. cerevisiae (Fig. 1). Candidacidal activity of Hst 5 was compared with the activities of Hst 3, Hst M, Hst 4, Hst 4a, and Hst C. Hst 5 exhibited similar candidacidal activity as Hst M and was 2.5- and 5-fold more potent than Hst 4 and Hst 3 (LD₅₀ = 1.8 µM versus 4.7 and 9.2 µM, respectively) (Table I). Hst 4a, which included three additional amino acids at its N terminus (GYK), had equivalent candidacidal activity with Hst 4. Hst C, which represents the eight-amino acid C terminus of Hst 3, did not produce loss of yeast cell viability even at high concentrations (500 µM). These data demonstrate that the middle and N-terminal regions of full-length Hsts are required for optimal candidacidal activity and that the C-terminal region may not be essential for yeast killing and may even reduce Hsts' fungicidal activity.

View this table: [in this window] [in a new window]   Table I Candidacidal activity of synthetic histatins Candidacidal assays were performed on C. albicans cells in the presence of Hst 5, Hst 3, Hst M, Hst 4, and Hst C (0.98-125 µM) and on spheroplasts in the presence of Hst 5 (0.98-250 µM), as described under "Experimental Procedures." Loss of viability is expressed as [1  (colonies after peptide addition/colonies after incubation in buffer only)] × 100. Results represent means ± S.D. of duplicates from 11 (H5, whole cells), 3 (Hst 5, spheroplasts), and 2 (Hst M, Hst 3, Hst 4, Hst C) experiments. Statistical significance was calculated using Student's t test for data from Hst 5-treated whole cells compared to loss of cell viability induced by Hst 4 (*, p < 0.05), Hst 3 (***, p < 0.005) and Hst 5 induced loss of spheroplast viability (***, p < 0.005). LD50, peptide concentration (µM) required to induce half-maximal loss of viability.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Hst 5-induced killing of C. albicans whole cells and spheroplasts and S. cerevisiae cells. C. albicans whole cells () and spheroplasts () or S. cerevisiae cells (, TI#20; , BJ2169) were incubated for 1.5 h at 37 °C with Hst 5 (0.98-125 µM) as described under "Experimental Procedures." Loss of viability is expressed as [1  (colonies after Hst 5 addition/colonies after incubation in buffer only)] × 100. Each data point is the mean of duplicate determinations from 3-11 independent experiments (± S.D.).

View this table: [in this window] [in a new window]   Table II Specificity of total 125I-Hst 5 binding and candidacidal activity C. albicans cells were incubated with 100 nM 125I-Hst 5 in the presence or absence of Hst 5, Hst 3, and Hst 4 (all at 100 µM) or 500 µM -amylase, insulin--chain, cytochrome c, or MUC7-derived peptides. Total binding is expressed as a percentage of control (average control was 1.9 ± 0.7 pmol/106 cells) and values are means ± S.D. from triplicates from five (cytochrome c) and three (Hst 5, Hst 3, Hst 4, -amylase, insulin -chain) and two (MUC7-derived peptides a and b) independent experiments. Candidacidal activity of Hst 5, Hst 3, and Hst 4 (125 µM), or -amylase, insulin -chain, and cytochrome c (500 µM), was assayed as described for Table I. Candidacidal results represent means ± S.D. from duplicates from 11 (Hst 5), 3 (-amylase, insulin -chain, cytochrome c), and 2 (Hst 3, Hst 4) independent experiments.

Hsts Induce Loss of Intracellular Dye from Yeast Cells-- Preincubation of C. albicans with a highly enriched fraction of histidine-rich polypeptides (HRPs) purified from human parotid saliva has been shown to produce rapid release of potassium from cells, thus implicating damage of the cell membrane as an early event in the HRP-induced killing (33). We examined the ability of synthetic Hsts to induce yeast cell membrane destabilization by measuring the release of the fluorescent dye calcein from yeast cells. C. albicans cells were loaded with calcein-AM, and fluorescent emission of the released free dye in response to Hsts was recorded over a period of 90 min.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Time course of Hst 5-induced calcein release from C. albicans whole cells and spheroplasts and killing of C. albicans whole cells. Calcein release measurements: C. albicans (106) cells were loaded for 2 h with 5 µM calcein-AM and were used as either untreated (whole cells) or converted to spheroplasts with Zymolyase in the presence of 2-mercaptoethanol as described under "Experimental Procedures." Whole cells were incubated with Hst 5 (50 µM, open circles; 200 µM, open squares), and spheroplasts were treated with Hst 5 (200 µM, open triangles) in spheroplast buffer. The fluorescent intensity of Hst 5-induced calcein release (Ir) was recorded every 5 min for 60 or 90 min at excitation and emission wavelengths of 485 and 530 nm, respectively. Calcein efflux was calculated as percent fluorescent recovery = [(Ir  Ibkg)/(Iint  Ibkg)] × 100, where Ibkg is fluorescent intensity measured before the addition of Hst 5 and Iint is fluorescent intensity corresponding to 100% of potentially available calcein determined by boiling of the cells. Each experimental point represents mean ± S.D. from three independent experiments. Time course of Hst 5-induced killing of C. albicans whole cells: C. albicans whole cells were incubated for the indicated periods at 37 °C with 31.25 µM Hst 5 (closed circles) as described under "Experimental Procedures." Loss of viability is expressed as [1  (colonies after Hst 5 addition/colonies after incubation in buffer only)] × 100. Each data point is the mean of duplicate determinations from three independent experiments (± S.D.).

Hsts Do Not Induce Leakage from Phospholipid Vesicles-- Candidacidal and dye release assays suggested that C. albicans components in the cell envelope are required for optimal Hst activity. To determine whether Hsts can disturb a lipid bilayer that does not contain proteins, model membranes of differing compositions were constructed. The lysis of lipid vesicles was monitored by the dequenching of fluorescence caused by leakage of encapsulated calcein, which is self-quenched inside liposomes. Bac 5, a proline- and arginine-rich polypeptide of bovine neutrophil granules with bactericidal activity against Gram-negative bacteria, was also tested in the dye release assay. We have previously shown that Bac 5 is a potent candidacidal molecule and exhibits lytic effects on negatively charged liposomes (23). Increasing concentrations of Hsts and Bac 5 were mixed with calcein-containing small unilamellar vesicles of varying phospholipid composition. Membrane disruption or perturbation was measured by fluorescent intensity of dye release. Bac 5 caused rapid dye release from ePC liposomes; however, neither Hst 5 nor Hst M elicited significant increase in fluorescence when mixed with ePC liposomes at molar ratios examined (Fig. 3). No release of dye was detected with any Hst when mixed with ePC liposomes containing ergosterol or cholesterol, or PS+cholesterol. These data point to differing mechanisms of candidacidal action between Hst and Bac 5. Bactenecins have been shown to effect Gram-negative bacteria by increasing the permeability of inner and outer membranes and disrupting membrane potential (50, 51). In contrast, salivary Hsts do not interact spontaneously with the model membranes selected, in agreement with previous conformational studies of Hst M (37), and suggest that a protein or other cellular components may be crucial for Hst killing ability.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Peptide-induced release of calcein from ePC liposomes. Small unilamellar vesicles containing calcein were prepared using reverse-phase evaporation procedures. Hst 5, Hst M, or Bac 5 were added to the liposomes at increasing peptide to lipid ratios, and the fluorescent intensity of the release dye (Ir) was recorded at excitation and emission wavelengths of 485 and 530 nm, respectively. Liposome preparation was lysed with 0.5% Triton X-100 to establish total fluorescent content, and percent fluorescent recovery was calculated as described in Fig. 2.

Analysis of Hsts Binding to C. albicans Cells-- To test whether C. albicans express binding sites for Hsts, binding experiments were performed using ¹²⁵I-Hst 5. Preliminary results were carried out to determine whether the iodinated Hst 5 retained biological activity. In a candidacidal bioassay, ¹²⁷I-Hst 5 in 10 mM phosphate buffer containing 2 mg/ml BSA had similar biological activity as the non-iodinated protein (data not shown).

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View larger version (17K): [in this window] [in a new window]   Fig. 4.   125I-Hst 5 binding to C. albicans cells. C. albicans (1 × 106 cells) were incubated for 20 min at room temperature with increasing concentrations of 125I-Hst 5 (800 pM to 2.4 µM). Specific binding (pmol/106 cells) was defined as the difference between total and nonspecific binding. Data are from a representative experiment and each point is the mean ± S.E. of triplicates. Scatchard analysis (inset) indicated 8 × 105 sites/cell with a dissociation constant (Kd) of 7.2 × 107 M. B is bound, and B/F is the ratio of bound to unbound 125I-Hst 5. Similar results were obtained in four additional experiments.

Specificity of ¹²⁵I-Hst 5 Binding-- Binding specificity was investigated using unlabeled Hst 5 and two related family members possessing candidacidal activities, Hst 3 and Hst 4, to compete with ¹²⁵I-Hst 5 for the identified sites on C. albicans. Hst 3 and 4 competed for the same binding sites as ¹²⁵I-Hst 5 with affinities (K_i = 8.4 ± 3.6 × 10⁷ M, n = 4 and 1.1 ± 0.28 × 10⁶ M, n = 2, respectively) which were similar to the affinity of ¹²⁵I-Hst 5 (K_D = 9.5 ± 4.9 × 10⁷ M) (Fig. 5). Moreover, the affinity of unlabeled Hst 5 (K_i = 7.8 ± 1.4 × 10⁷ M, n = 2) was very similar to the affinity of ¹²⁵I-Hst 5, verifying that radiolabeled Hst 5 retained full binding ability. Although candidacidal assays showed differences in the potency of Hst 5, Hst 3, and Hst 4, data from competition binding experiments indicated these three Hsts have approximately the same affinities to the ¹²⁵I-Hst 5 sites on C. albicans. We next tested whether salivary amylase, insulin, and cytochrome c, which were used in candidacidal assays, interact with binding sites on C. albicans (Table II). Even at high concentrations, salivary amylase and insulin did not produce a significant decrease in the total ¹²⁵I-Hst 5 binding. In addition, cytochrome c, which induced 50% loss of C. albicans cell viability, did not inhibit ¹²⁵I-Hst 5 binding.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Inhibition of 125I-Hst 5 binding by Hst 5, Hst 4, and Hst 3. C. albicans (1 × 106 cells) were incubated for 20 min at room temperature with 125I-Hst 5 (100 nM) and various concentrations (50 nM to 500 µM) of unlabeled Hst 5 (closed squares), Hst 4 (open circles), or Hst 3 (open squares). The results are expressed as the percentage of total 125I-Hst 5 bound in the absence of competitor and are means ± S.D. of triplicates from four (Hst 3) and two (Hst 5 and Hst 4) separate experiments. The curves were calculated from a one-site competitive inhibition equation. Binding constants (Ki) for Hst 5, Hst 4, and Hst 3 were determined as 7.8 ± 1.4 × 107 M, 1.1 ± 2.8 × 106 M, and 8.4 ± 3.6 × 107 M, respectively.

Identification of a C. albicans Hst 5-binding Protein-- We next examined whether Hst 5 binds to C. albicans proteins using an overlay assay. C. albicans cells were disrupted with glass beads and proteins from whole cell lysates and crude membrane fractions were separated by gel electrophoresis and analyzed on nitrocellulose membranes with radiolabeled Hst 5. The results revealed that ¹²⁵I-Hst 5 bound to a C. albicans protein with an apparent molecular mass of 67,000 ± 4000 (n = 4). This protein was detected in C. albicans whole cell lysate and crude membrane fraction and was the only protein consistently observed to bind Hst 5 (Fig. 6, lanes 1 and 2). In some experiments, a similar sized protein was found in the high speed supernatant containing the soluble post-membrane fraction (data not shown). ¹²⁵I-Hst 5-binding proteins were not detected from Zymolase C. albicans cell wall digest or lysates prepared from human embryonic kidney epithelial (HEK 293) cells or neutrophil plasma membranes, even in longer exposures (data not shown). Interestingly, when proteins from Hst 5-susceptible yeast S. cerevisiae strain TI#20 were incubated with ¹²⁵I-Hst 5 on overlay assay, a doublet with relative molecular masses of 70,000 and 87,000 was observed (Fig. 6, lane 3). The faster migrating component was of similar size and may be related to the C. albicans Hst 5 binding component, or alternatively, may be a degradation fragment of the slower migrating protein in S. cerevisiae.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Overlay assay of the binding of 125I-Hst 5 to proteins from yeast cells. C. albicans and S. cerevisiae (strain TI#20) were disrupted with glass beads and whole cell lysates and crude membrane fractions were prepared as described under "Experimental Procedures." Solubilized proteins were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated for 4 h with 50 nM 125I-Hst 5 in binding buffer. Lane 1, C. albicans whole cell lysate; lane 2, C. albicans crude membrane fraction; lane 3, S. cerevisiae strain (TI#20) whole cell lysate. The molecular sizes (× 103) of protein standards are indicated by bars to the left and the position of 125I-Hst 5-binding proteins by arrows.

View larger version (66K): [in this window] [in a new window]   Fig. 7.   Cross-linking of 125I-Hst 5 to C. albicans cells. C. albicans cells were incubated for 20 min at room temperature with 125I-Hst 5 in the presence (lane 2) or absence (lanes 1 and 3) of excess of unlabeled Hst 5. Cells were then placed on ice and incubated for 20 min with 0.9 mM non-cleavable membrane-permeable cross-linking reagent DSS. Cells were processed as described under "Experimental Procedures," and cell lysates (lanes 1 and 2) or cell wall material (lane 3) were subjected to SDS-PAGE and analyzed by autoradiography. Bars indicate the position of molecular size protein standards (× 103), and the arrow indicates the cross-linked complex.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The family of salivary Hsts consists of structurally related, low molecular weight histidine rich proteins, which are part of the non-immune host defense system of the oral-esophageal area. The finding that Hsts are potent in vitro antifungal agents, while nontoxic to human cells, provided promise for their therapeutic potential in patients with oral candidiasis. Despite the characterization of Hsts at the structural and conformational level (38), at present little is known about their fungicidal mechanism of action and the basis for selectivity between eukaryotic fungal and host mammalian cells. This study represents the first description of Hst 5 binding sites on C. albicans that mediate cell killing, and identifies a 67-kDa yeast Hst 5-binding protein. We report here that Hst-induced killing is initiated upon binding to a candidal receptor rather than spontaneous permeabilization and destabilization of cell membranes based on biological and fluorescent dye release assays as well as Hst 5 binding and cross-linking studies. Hsts binding to a specific component on target yeast cells may be the basis for their selectivity and lack of toxicity to human host cells.

This study used three major salivary Hsts and the middle active region (Hst M), which represent truncated variants of full-length salivary Hst 3. The candidacidal activity of the synthetic peptides, shown here, is consistent with the reported activities of naturally occurring (6, 11, 15), synthetic (17), and recombinant (18, 36) salivary Hsts. Presence of the C-terminal portion of the molecule reduced the fungicidal activity, as demonstrated by the lower activity of Hst 3 and Hst 4 when compared with structurally equivalent Hst 5 and Hst M, which lack the C-terminal region (Table I). Thus, full-length Hst 3 may be a precursor form of Hsts and proteolytic cleavage of the C-terminal region represents a post-translational processing step for activation of the protein. This concept is in agreement with the findings of Perinpanayagam et al. (14) that Arg²⁵ is a primary cleavage site of Hsts in vivo. This cleavage site resides at the junction of M and C regions defined in the present study, and may have functional significance in proteolytic processing to produce a functional molecule. In contrast, proteolytic cleavage at other sites in the defined M and N-terminal regions may produce a less active Hst. We have shown that C. albicans cells have the ability to degrade ¹²⁵I-Hst 5 (45). In this respect, less active or inactive Hst fragments detected in vivo (14) and generated by proteolytic cleavage at sites Lys⁵, Arg⁶, Lys¹², His¹⁸, and Arg²² may represent a microbial protective mechanism against the cytotoxic Hsts.

Many polypeptide antibiotics exert their activity through spontaneous insertion into microbial membranes forming ion channels or pores, thereby disrupting cell structure and integrity. If Hsts function primarily as membrane lytic agents, direct exposure to the plasma membrane of wall-free spheroplasts should enhance Hst candidacidal activity. However, treatment of C. albicans cells with Zymolyase in the presence of a reducing agent to generate wall-free cells resulted in a 14-fold reduction of cellular sensitivity to Hst 5 compared with intact cells (Table I and Fig. 1). Zymolyase-20T preparation contains a mixture of hydrolytic enzymes including -1,3-laminaripentaohyhydrolase, -1,3-glucanase, protease, and mannase (52). Therefore, either the yeast cell wall is necessary for Hst killing, or an enzymatic cleavage and/or alteration of specific yeast membrane component critical for optimal killing accounts for decreased sensitivity of spheroplasts to Hst 5. Similarly, Lactococcus lactis spheroplasts or trypsin-pretreated whole cells were less sensitive to the bacteriocin Lactostrepcin 5, thus implicating a putative membrane receptor mediating Lactostrepcin 5 and Lactococcin A effects (53). Driscoll and colleagues (54) found that lyticase treatment of C. albicans in the presence of 2-mercaptoethanol resulted in spheroplasts equally sensitive to Hst 5 as the intact cells. However, these lyticase-produced spheroplasts survived a 30-min incubation in hypotonic 10 mM potassium phosphate buffer, which should lyse osmotically sensitive cells. Different methodologies used for generating spheroplasts may explain apparent differences in the sensitivity of Zymolyase- and lyticase-produced spheroplasts to Hst 5 in this study.

A wide variety of naturally occurring peptide antibiotics have been identified and isolated, including defensins and bactenecins from neutrophils, maganins from frog skin, tachyplesins and polyphemusins from hemocytes of horseshoe crabs, and melittin from bee venom (19). Despite structural variations, virtually all carry a net positive charge and their antimicrobial activities are related to the ability to disrupt microbial membranes. Bactenecins, defensins, maganins, and tachyplesins have been shown to adopt amphiphilic -helical, poly-L-proline II, or -sheet structures leading to spontaneous insertion into membrane and disruption of cell structure (23, 55). In contrast, structural and conformational analysis of Hst 5 revealed that the weak amphipathic character of the helical structure precludes spontaneous insertion into microbial membranes and formation of pores or ion channels across the membrane (37, 38). Hsts' inability to disrupt lipid membranes, measured here by the release and dequenching of calcein, further supports these observations. Unlike Bac 5, which was effective in disrupting artificial membranes (Fig. 3) (23), Hst 5 and Hst M do not display membrane lytic activities to any of the membranes selected. Furthermore, analysis of the magnitude and time course of Hst 5-induced calcein release from C. albicans cells (Fig. 2) showed that loss of cell integrity is a secondary effect following cell death rather than the result of primary disruption of the membrane by channel or pore formation. Together, these results suggest a selectivity of Hst fungicidal activity that is exerted through interaction with a specific yeast component(s). An early study utilizing a partially purified fraction of HRPs from human parotid saliva reported 90% release of potassium from C. albicans cells after a 30-min exposure to HRPs (33). Although this work correlated HRP-induced loss of cell viability with loss of cell membrane integrity, our calcein-release experiments utilizing pure synthetic Hst 5 indicate that cell death is not correlated with leakage of the dye, e.g. with Hst 5-induced yeast plasma membrane disruption. The observed difference in the response of C. albicans cells to a mixture of purified HRPs reported previously (33) as compared with pure Hst 5 in the present study is difficult to explain. The use of purified mixtures of HRPs in the earlier studies suggests the possibility of the presence of additional salivary components in the mixture, which could be responsible for potassium leakage from the cells. Moreover, the correlation of candidacidal activity and the leakage of potassium ions from yeast cells using a mixture of Hsts that exhibit variations in their candidacidal potency and multifunctional properties may not be unequivocal.

¹²⁵I-Hst 5 equilibrium binding results indicated that C. albicans cells express a class of saturable binding sites for Hst 5, Hst 3, and Hst 4, numbering 8.6 × 10⁵/cell (Fig. 4). The affinity binding constant (K_d~1 µM) is in agreement with the active concentrations on the candidacidal assay (LD₅₀ = 1.8 µM) (Table I) and is similar to the K_d for ¹²⁵I-Hst 5 in equilibrium binding studies on Porphyromonas gingivalis (K_d = 1.5 µM) (56). Although Hst 5 bactericidal activity to P. gingivalis has not been reported, Hst 5 has been shown to be effective in inhibition of coaggregation of these cells with other bacteria by binding to specific sites numbering 3600/cell.

Analysis of Hst binding and killing of C. albicans spheroplasts further suggested a correlation between binding and killing ability. Specific ¹²⁵I-Hst 5 binding was not detected to spheroplasts, which were 14-fold less susceptible to killing, compared with intact cells (Table I). The appearance of lower affinity and/or a larger number of binding sites for Hst 5 not detected with the concentrations of ¹²⁵I-Hst 5 used in the whole cell binding assays could explain our inability to measure specific Hst 5 binding to spheroplasts. Hst 3 and Hst 4 recognized ¹²⁵I-Hst 5 sites on C. albicans with very similar affinities (Fig. 5), although they were 5- and 2.5-fold, respectively, less potent than Hst 5 on the candidacidal assays (Table I). Observed differences in candidacidal activities of Hst 3, Hst 4, and Hst 5 may reflect different biological effects including killing, inhibition of germination, or increased proteolysis. We selected three proteins for use in our killing and binding assays to address the specificity of Hst 5 candidacidal and binding activities: -amylase, a salivary protein; insulin--chain with a similar size; and cytochrome c, which has a high pI. Amylase and insulin neither induced cell killing nor competed for Hst 5 binding sites (Table II). Cytochrome c (500 µM) did not inhibit ¹²⁵I-Hst 5 binding; however, it induced about 50% loss of C. albicans cell viability, thus suggesting a different mechanism for Hst and cytochrome c yeast cell killing. Earlier experiments have shown that cytochrome c exhibits lytic activity to C. utilis, perhaps due to electrostatic interactions with the yeast cell membrane (47). Recent studies have indicated an involvement of cytochrome c in apoptosis of eukaryotic cells by its release from the mitochondrial intermembrane space into the cytosol (57).

Overlay assays demonstrated the existence of a candidal 67-kDa ¹²⁵I-Hst 5-binding protein (Fig. 6). This protein was detected in whole cell lysates and crude membrane fractions prepared from glass-bead disrupted C. albicans cells, but not in the yeast cell wall fraction. Moreover, cross-linking experiments revealed the presence of a specific 73-kDa ¹²⁵I-Hst 5-containing complex (Fig. 7). This complex may contain one or two molecules of ¹²⁵I-Hst 5 covalently linked to the 67-kDa binding protein, detected in the overlay assays. Because of the small size of Hst 5 (~3 kDa), our experiments did not clarify the exact stoichiometry of the binding event. The absence of cross-linked products in the cell wall material further confirmed overlay results that the Hst 5-binding protein is not located on the cell wall. ¹²⁵I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20 (Fig. 6), but was not detected in extracts from human kidney epithelial cells and neutrophils, suggesting that the presence of a yeast receptor for Hst may be the basis for its lack of toxicity to human cells. The presence of an additional higher molecular mass binding protein in S. cerevisiae TI#20 cells may account for the differences in the sensitivity of C. albicans and S. cerevisiae TI#20 to Hst 5. Interestingly, preliminary overlay assay data showed that Bac 5 partially competed with labeled Hst 5 for the C. albicans-binding protein.³ Although Bac 5 differs from Hst 5 in its ability to lyse lipid membranes, it is possible that its killing mechanism may overlap that of Hst 5. Additional experiments are needed to support this assertion.

Although the exact location of the Hst 5-binding protein is yet to be determined, our data show that it is not located within the yeast cell wall. Removal of the cell wall with 2-mercaptoethanol and Zymolyase, a preparation containing a variety of hydrolytic enzymes including a protease, produced cells with decreased Hst 5 sensitivity, suggesting alteration of a cell membrane protein as the source of reduced Hst 5 effect. Preliminary cross-linking experiments that employed membrane-impermeable cross-linking reagent BS to covalently link ¹²⁵I-Hst 5 to C. albicans cells detected a ¹²⁵I-Hst 5-containing complex of similar size (data not shown) to the one formed with its membrane-permeable analog DSS (Fig. 7). These results, together with the detection of Hst 5-binding protein in the C. albicans crude membrane fraction (Fig. 6, lane 2), provide evidence that Hst 5-binding protein may be located on the plasma membrane.

C. albicans has been shown to express several adhesins that are receptors for fibronectin, fibrinogen, laminin, collagen, and complement receptors for C₃d and iC₃b (1, 58-62). These receptors uniformly have a yeast cell wall location and are associated with pathogenicity of Candida species as related to adherence to epithelial and endothelial tissues. It has been suggested that similarities between these receptors may in fact reflect a single C. albicans surface mannoprotein with a number of binding activities (59). The cell wall location and binding characteristics of these candidal mannoprotein receptors (K_d ~1 nM, numbering about 5000/cell) differ from the presently characterized Hst 5-binding protein. Hst binding characteristics reported here are more similar to those described for the pore-forming colicins (63). These bacterial toxins kill susceptible Escherichia coli by formation of ion channels in the inner membrane of the target cells via binding to specific outer membrane receptors. The density of OmpF, the receptor for colicin N, has been evaluated at approximately 4 × 10⁵ copies/E. coli with a binding affinity K_d~1 µM (64, 65). Following binding, colicins are translocated across the cell envelope by helper proteins, which belong to nutrient uptake systems.

It is currently unclear how association of Hsts with the yeast-binding protein leads to cell death. Hsts may utilize an existing yeast membrane transport system for vital nutrients to gain illicit access into the cell where secondary effects occur. Membrane-bound transport proteins have been described for both C. albicans and S. cerevisiae, which are involved in uptake of amino acids, peptides, and sugar molecules (66, 67). Yeasts also express drug efflux pumps which have been divided into two major classes: membrane proteins belonging to the ABC (ATP binding cassette) or MSF (major facilitator superfamily) families, which share structural and presumably functional homology to bacterial components involved in the active transport of a wide range of molecules including oligopeptides, histidine, maltose, ribose, phosphate, vitamin B₁₂, and hemolysin) (68, 69). Intracellularly, Hsts may effect sites such as mitochondria to inhibit energy production, induce release of cytochrome c, a process characteristic for apoptosis, or inhibit DNA or RNA synthesis. Finally, interaction with the yeast receptor may be required for correct orientation or oligomerization of Hst following insertion into the membrane. In this respect, -hemolysin, a toxin secreted from the human pathogen Staphylococcus aureus binds to the membrane as a monomer, forms a heptamer and then inserts and lyses human platelets, erythrocytes, and endothelial cells (70).

In conclusion, major disadvantages of currently used antifungal drugs are their toxicity and the development of resistant yeast strains (71). In contrast, salivary Hsts are nontoxic to humans and yet potent candidacidal agents even with drug-resistant strains. Although the therapeutic potential of Hsts is becoming apparent, particularly against azole-resistant strains, the exact mechanism of cell killing must be elucidated to fully utilize them as therapeutic agents. The identification of a specific Hst 5-binding protein on C. albicans in this study provides insight into the potential mechanism of yeast killing and suggests a basis for the nontoxic nature of Hsts. Finally, the binding characteristics of Hst 5, described here, are in agreement with the observed potency of its biological effect and further provide crucial information to the use of Hst 5 as a therapeutic agent.