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J Biol Chem, Vol. 274, Issue 27, 18872-18879, July 2, 1999


Salivary Histatin 5 Induces Non-lytic Release of ATP from Candida albicans Leading to Cell Death*

Svetlana E. KoshlukovaDagger , Tracy L. LloydDagger , Marcelo W. B. AraujoDagger , and Mira EdgertonDagger §

From the Departments of Dagger  Oral Biology and § Restorative Dentistry, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5'O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oral candidiasis is a superficial mucosal infection in which Candida albicans is the predominant species isolated from infected areas (1). Pathogenicity of C. albicans is postulated to involve adhesion to host cells and filamentous growth (2). Incidence of oropharyngeal candidiasis has risen dramatically in the past 20 years due to increased antibiotic and pharmaceutic drug use and longer survival of people with compromised immune systems including patients on cancer chemotherapy, diabetics, and premature infants. Oropharyngeal candidiasis is so frequently associated with AIDS that it is a criterion for staging progression of disease (3). Increased use of current antifungal agents to treat candidiasis in late stage AIDS and cancer patients has resulted in emergence of Candidal species with antifungal drug resistance, especially to azole-based drugs. Even with aggressive treatment with the available antifungal drugs, morbidity from C. albicans infections in immunocompromised patients is high. These facts point to a pressing need for new antifungal agents and improved drug therapies.

Innate host defense systems include a wide variety of small (3000-5000 Da) cationic proteins with potent antibacterial and antifungal activity (4). Most locations that are in contact with indigenous microorganisms directly express or posses cells that can produce antimicrobial proteins. Despite structural variations, virtually all antimicrobial proteins carry a net positive charge.

Histatins (Hsts)1 are structurally related histidine-rich basic proteins of acinar cell origin expressed only in humans and higher subhuman primates (5, 6). Salivary Hsts possess in vitro candidacidal and candidastatic activities (5, 7), and to a lesser degree bactericidal properties (8). Hst 1 and Hst 3 are the full-length proteins (38 and 32 amino acids, respectively) encoded by two closely linked and related genes, HIS1 and HIS2 (9), while Hst 2 and Hsts 4-12 are generated by proteolytic cleavage during secretion (10). In vitro, Hst 5 (24 amino acids) is the most potent candidacidal member of the family that kills yeast and filamentous forms of Candida species at physiological concentrations (15-50 µM) (11-13). Salivary Hsts have potential as therapeutic agents in patients with oral candidiasis, being potent antifungal agents, while non-toxic to humans.

Hsts have similar size and net positive charge as other naturally occurring antimicrobial proteins; however, they possess unique structural features such as high histidine content and lack of disulfide bonds. Extensive structural and conformational analysis of Hst 5 revealed that the weak amphipathic character of the helical structure precludes spontaneous insertion into microbial membranes and direct formation of pores or ion channels across the membrane (12, 14). Furthermore, Hst 5 variants with reduced killing ability exhibited similar helical content to Hst 5, suggesting the alpha -helical conformation alone is not solely responsible for optimal candidacidal activity (15). In contrast to the wealth of knowledge obtained on the primary structure and conformation of Hst 5, very little is known about its mode of action. Previous studies on the biochemical mechanism of Hsts activity have revealed three major findings: 1) K+ was rapidly released following exposure of C. albicans cells to a mixture of purified Hsts (16); 2) Mg2+ and Ca2+ inhibited Hst 5-induced killing of C. albicans (11); 3) exposure of C. albicans cells for 1 h to Hst 1 did not alter membrane permeability to the vital dyes methylene blue and acridine orange (13). Although altered yeast membrane permeability would readily account for the observed release of K+, Hst 5 direct channel-forming properties or Hst-induced nonspecific permeabilization of C. albicans cell membrane is not supported by the structural studies (14), early dye exclusion assays (13), or Hsts' inability to lyse lipid membranes (17). We have recently reported the presence of functional C. albicans-binding sites for salivary Hsts and a 67-kDa yeast Hst 5-binding protein. This finding provided a new insight into the potential mechanism of killing and suggested a basis for the selectivity of Hst 5 yeast killing and lack of toxicity to human host cells (17).

The physiological mechanism of action of a number of cationic host defense proteins including bactenecins (18) defensins (19, 20), cecropins (21), and maganins (22) is thought to be related to their abilities to disrupt lipid packing or form ion-permeable channels (14, 15). The currently used drugs in treatment of candidiasis, polyene antimycotics and azole derivatives have also been reported to alter yeast membrane permeability. Polyene antimycotics complex with ergosterol of the plasma membrane resulting in release of cellular K+ (23, 24). The azole-based drugs inhibit the biosynthesis of ergosterol (23, 25) and like the polyene antibiotics and some antimicrobial proteins, induced release of K+ and 260 nm absorbing materials from C. albicans cells (26). The ultraviolet absorbing materials could represent ATP since release of cellular ATP was detected following 10 min of C. albicans treatment with ketoconazole and other azole derivatives (27). Cell lysis is one route by which ATP could be released from the cells. However, release of cytosolic ATP via plasma membrane channels in the absence of cytolysis has also been described, and members of the ATP-binding cassette (ABC) protein family were putatively associated with conductive ATP transport (28). Furthermore, released ATP can function outside of the cell via activating membrane purinergic receptors of the P2X family to cause increased K+ and Cl- membrane permeability and even cell lysis (29-31).

Hst 5 has been characterized at structural and conformational levels with the aim of producing more effective fungicidal protein. However, despite extensive efforts, this goal has not been achieved in large part because of lack of understanding of the cellular target and mechanism of action of Hsts. We examined the biochemical mechanism of Hst 5 yeast killing by performing pharmacological assays and functional studies on C. albicans cells. We showed that Hst 5 induced efflux of cellular ATP that correlated with cell death. Furthermore, the detected extracellular ATP did not represent leakage from lysed cells, but rather appeared to be released from intact and metabolically active cells. We conclude that, under the experimental paradigms used, extracellular ATP released by Hst 5 can activate putative purinergic receptors on C. albicans to induce cell death.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- C. albicans strain DS1 was isolated from the palate of a denture stomatitis patient (12) and strain 31531A was obtained from Dr. E. Rustashenko and Dr. F. Sherman, Department of Biochemistry and Biophysics, University of Rochester. Sabouraud dextrose agar and YPD media were from Difco (Detroit, MI). Actinomycin D, tunicamycin, staurosporine, verapamil, oligomycin, and valinomycin were purchased from Calbiochem, and cyclohexamide, N-ethylmaleimide, 2-deoxyglucose, orthovanadate, antimycin A, salicylhydroxamic acid, CCCP, DNP, azide, suramin, PPAD, BzATP, and ATPgamma S were from Sigma. DiOC5(3) was purchased from Molecular Probes (Eugene, OR).

Hst 5 Synthesis and Purification-- Hst 5 (DSHAKRHHGYKRKFHEKHHSHRGY) was synthesized using a Beckman System 990 synthesizer, standard solid phase synthesis protocols and N-(9-fluorenyl) methoxycarbonyl chemistry as described previously (17). The protein was cleaved from the resin and purified by reversed phase high performance liquid chromatography using a Rainin Dynamax-60A C18 column and a gradient of acetonitrile and water (both containing 0.1% trifluoroacetic acid) as the solvent system. Purity of Hst 5 was assessed by amino acid analysis, mass spectroscopy, and confirmed by amino acid sequencing using an Applied Biosystems (model 471A) protein sequencer.

Candidacidal Assay-- C. albicans was maintained on Sabouraud dextrose agar and grown in synthetic medium as described previously (17). Antifungal activity of Hst 5 was examined by microdilution plate assay (17) with the following modifications. Briefly, C. albicans cells were washed twice with 10 mM sodium phosphate buffer (Na2HPO4/NaH2PO4), pH 7.4, and resuspended at 5 × 105 or 5 × 106 cells/ml. Cell suspensions were mixed with the indicated pharmacological agents and incubated for 2 h (unless indicated otherwise) at 37 °C with shaking. CCCP was dissolved in methanol and freshly made stock solutions of DNP were prepared in dimethyl sulfoxide and diluted prior to use (the final concentration of methanol and dimethyl sulfoxide did not exceed 1%). Control cultures were incubated with 20 µl of 10 mM phosphate buffer or vehicle (1% dimethyl sulfoxide or methanol) alone. Cells were then left untreated or treated with Hst 5 (final concentration 31 µM) for an additional 1.5 h. Cell suspensions were diluted and aliquots (250 cells) were spread onto Sabouraud dextrose agar plates and incubated for 24 h at 37 °C. The optimal concentrations of pharmacological agents and solvents were determined in preliminary experiments to avoid potential artifacts from toxic effects. Candidacidal assays were performed in duplicate or triplicate. Cell survival was expressed as percentage of control and loss of viability was calculated as (1 - (colonies from Hst 5-treated cells/colonies from control cells)) × 100.

ATP Bioluminescence Assay-- ATP levels in cultures of C. albicans were measured as described (27, 32) with the following modifications. For intracellular ATP measurements: 105 C. albicans cells were mixed with CCCP (500 µM), DNP or azide (both at 10 mM) to a final volume of 200 µl and incubated 3.5 h at 37 °C with shaking. In some experiments cells were treated with CCCP, DNP, or azide for 2 h at 37 °C, followed by the addition of Hst 5 (final concentration of 61 µM) and the incubations were carried out for 1.5 h at 37 °C with shaking. Control cultures were incubated with 10 mM phosphate buffer or vehicle only. For the time course and dose-dependence experiments, cells were treated with either 61 µM Hst 5 for various times, or with increasing concentrations of Hst 5 for 1.5 h. Cells were harvested by centrifugation (5000 × g, 5 min), washed with TE buffer (50 mM Tris, 2 mM EDTA, pH 7.8), and cell pellets were submerged in liquid nitrogen followed by the addition of 2 ml of boiling TE. Cells were boiled for an additional 4 min, centrifuged, and cell lysates were placed on ice until assayed for ATP. For extracellular ATP measurements: cells were pelleted following incubations and 50 µl of the supernatant was pipetted into 450 µl of boiling TE, boiled for additional 90 s, and stored on ice until assayed for ATP. Extracellular and intracellular ATP levels were measured by luminometry using an ATP Assay Kit (Sigma) according to manufacturer's instructions. Luciferin-luciferase assay mixture (100 µl) was added to 100 µl of cell lysates or 50 µl of extracellular material in 96-well microtiter plates and light emission was monitored in a 1250 LKB-Wallac luminometer. Results are expressed in bioluminescence relative light units (RLU) and ATP concentrations determined from ATP standard curves.

Flow Cytometry-- C. albicans (106 cells, to permit acquisition of 105 events by FACSCAN) were mixed with CCCP (500 µM), DNP or azide (both at 10 mM), or Hst 5 (61 µM) to a final volume of 250 µl and incubated for the indicated periods at 37 °C with shaking. The membrane potential sensitive fluorescent dye DiOC5(3) (33, 34) was added for 10 min (0.5 µM final concentration) and samples analyzed on a FACSCAN flow cytometer (Becton Dickinson) using a 15 mW argon laser at 488 nm excitation and sideward light scatter (SSC, 340 PMT) and fluorescence (500 PMT). Data were recorded as histograms of fluorescence (FL1) versus counted events.

Cell Respiration Measurements-- Oxygen consumption was measured using a Clark type oxygen electrode (YSI 5300 Biological Monitor, Yellow Springs Instrument, Yellow Springs, OH). C. albicans cells (2 × 106) were treated for 10 min or 1.5 h at 37 °C in the presence or absence of 31 µM Hst 5. The cells were then transferred to the chamber of the electrode in a final volume of 2 ml of 10 mM phosphate buffer and oxygen consumption was measured for 10 min with stirring. In some experiments antimycin A (1 µg/ml) was added directly to the cells in the chamber. Oxygen uptake was expressed as nanomole of O2/min/106 cells.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemical Uncouplers Protect C. albicansCells from Hst 5 Killing-- We attempted to find pharmacological agents or treatments capable of selectively inhibiting Hst 5-induced killing of C. albicans in order to understand the biochemical basis of its candidacidal activity. A large number of agents were used at concentrations known to affect transcription, translation, protein glycosylation, kinases, ATPases, Ca2+ channels, glycolysis, mitochondrial energy production, and respiration (Table I). Of all agents tested, only the proton ionophore CCCP had an inhibitory effect on Hst 5-induced killing of C. albicans. Therefore, we exploited the ability of this agent to protect cells from Hst 5 killing to examine the mechanism of yeast cell death.

                              
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Table I
Agents that failed to inhibit Hst 5-induced killing of C. albicans (DS1)

Under the conditions employed, CCCP provided substantial protection to the cells against Hst 5 killing in a concentration-dependent manner, while not impairing the viability of control cells (Fig. 1). Cells preincubated with 500 µM CCCP (the highest concentration tested) exhibited about 50% increase in viability over that of untreated cells. CCCP was effective against the lethal action of Hst 5 on a different strain of C. albicans, 3153A, and afforded similar protection to the one observed with DS1 strain, thus suggesting that the CCCP effect was not strain specific (Fig. 1). The protective effects of CCCP were depended on the length of exposure to the cells prior to Hst 5 treatment. Preincubation of C. albicans cells for 10 min with 500 µM CCCP did not significantly reduce cell killing resulting from subsequent incubation for 1.5 h with Hst 5. Increased time of CCCP treatment caused a gradual increase (52% at 2 h) in cell survival (Fig. 2).


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  		Fig. 1.   CCCP-induced protection of C. albicans against Hst 5 killing. Candidacidal assays were performed on C. albicans strains DS1 and 3153A. Cells were incubated for 2 h at 37 °C with CCCP (10-500 µM) and were then left untreated or treated with 31 µM Hst 5 for 1.5 h and processed as described under "Experimental Procedures." Cell survival is expressed as percentage of control and values are mean ± S.D. from duplicates from four independent experiments. Control colony forming units (CFU) (230 ± 12) was average of CFU from cells incubated in 10 mM phosphate buffer (206 ± 18 CFU), 1% methanol (236 ± 15 CFU), 10 µM CCCP (241 ± 11 CFU), 100 µM CCCP (221 ± 16 CFU), and 500 µM CCCP (236 ± 28 CFU).


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  		Fig. 2.   Time course of CCCP-induced protection of C. albicans against Hst 5 killing. C. albicans cells, strains DS1 (closed circles) and 3153A (open circles) were incubated for the indicated periods at 37 °C in the presence of 500 µM CCCP and then treated with 31 µM Hst 5 for 1.5 h as described under "Experimental Procedures." In all cases cell survival is expressed as percentage of control and each data point is the mean ± S.D. of duplicate determinations from three independent experiments.

The nature of CCCP as a classical uncoupler of oxidative phosphorylation (35) prompted an examination of other uncouplers such as DNP and sodium azide (36, 37) for effects on Hst 5-induced killing of C. albicans. Neither DNP nor azide was candidacidal at the concentrations tested and provided protection to the cells from Hst 5 cytotoxic activity in a concentration-dependent manner. C. albicans cells preincubated for 2 h with 10 mM DNP (the highest concentration tested) and subsequently exposed to 31 µM Hst 5 exhibited about 69% (strain DS1) and 55% (strain 3153A) increase in viability over that of cells treated with Hst 5 only (Fig. 3). Azide at concentrations of 10 mM was the most effective agent in protecting DS1 cells from Hst 5 killing (82% increase of viability); 3153A cells were slightly less sensitive to azide compared with DS1 and maximal increase of viability was 66% (Fig. 3). This level of protection required more than 90 min of exposure of the cells from both Candida strains to DNP or azide, prior to Hst 5 treatment (data not shown). Together, these findings demonstrate that Hst 5-induced killing of C. albicans can be inhibited by three structurally different chemical uncouplers. The protective effects of CCCP, DNP, and azide may reflect their ability to dissipate proton gradients across cell membranes as well as to inhibit mitochondrial ATP production.


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  		Fig. 3.   Effects of DNP and azide on Hst 5 induced killing of C. albicans. C. albicans cells (strains DS1 and 3153A) were incubated for 2 h at 37 °C in the presence or absence of the indicated concentrations or DNP (A) or azide (B) and then treated with 31 µM Hst 5 for 1.5 h as described under "Experimental Procedures." Cell survival is expressed as for Fig. 1 and each data point is the mean ± S.D. of duplicate determination from four independent experiments.

Hst 5 Induces Release of Cellular ATP and Killing of C. albicans-- At the cellular level, one of the earliest effects of oxidative stress is dissipation of transmembrane cation gradients and subsequent depletion of ATP stores (38). To test whether the protective effects of CCCP, DNP, and azide were due to inhibition of energy-dependent interaction between Hst 5 and yeast cells, ATP content of C. albicans was measured by luminometry. For these experiments cell number, incubation time, and concentrations of the agents were maintained as in the candidacidal assay. CCCP (500 µM) and azide (10 mM) provided protection of 52 and 82% to DS1 cells against Hst 5 lethal activity (Figs. 1 and 3), however, neither of these agents significantly altered the ATP content of these cells (Fig. 4). This finding is consistent with a previous report that ATP pools in azole-resistant and -sensitive C. albicans cells were not reduced 30 min after incubation with CCCP and FCCP (39). DNP (10 mM) caused about 2-fold reduction in cellular ATP in agreement with earlier findings that DNP activates cellular ATPases (40). Therefore, the common protective effects of sodium azide and the proton ionophores CCCP and DNP cannot be explained by an inhibition of ATP production in mitochondria.


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  		Fig. 4.   Effects of the uncouplers and Hst 5 on C. albicans intracellular ATP. C. albicans cells (strain DS1) were incubated for 3.5 h at 37 °C with CCCP (500 µM), DNP or azide (10 mM), or cells were left untreated for 2 h and then exposed to 61 µM of Hst 5 for additional 1.5 h. Yeast cell lysates were prepared as described under "Experimental Procedures" and ATP was measured by luminometry. Results are expressed as percentage of intracellular ATP in control cells (incubated for 3.5 h in 10 mM phosphate buffer); 100% average was 1.1 ± 0.1 × 104 bioluminescence RLU. Each data point represents mean ± S.D. from duplicates from three independent experiments.

Surprisingly, Hst 5 treatment of C. albicans cells alone resulted in a drastic reduction of cellular ATP content. In C. albicans cells exposed for 1.5 h to Hst 5 intracellular ATP was decreased to 6% of control (Fig. 4). The effect of Hst 5 on ATP depletion was observed as early as 10 min of treatment and was dose dependent over the same concentrations of Hst 5 active in the candidacidal assay (data not shown). In order to investigate the cause of the observed intracellular ATP depletion, we designed experiments to quantify extracellular ATP levels. An increase in extracellular ATP was detected following Hst 5 treatment (Fig. 5) which coincided with the time of Hst 5-induced depletion of intracellular ATP. Furthermore, concentrations of Hst 5 effective in the candidacidal assays and in depletion of intracellular ATP caused an 180-fold increase in extracellular ATP (Fig. 5). The extracellular ATP detected after 1.5 h of exposure of the cells to Hst 5 represented approximately twice the amount detected following 10 min of exposure (4.6 × 106 and 2.5 × 106 RLU). Our ATP release assays were routinely performed with 5 × 105-106 cells and the detected extracellular luminescence after Hst 5 treatment of the cells corresponded to approximately 0.4 fmol of ATP released/cell. This is about half of the reported C. albicans intracellular ATP pool concentration (0.75 fmol/cell) (39).


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  		Fig. 5.   Hst 5-induced ATP release and killing of C. albicans. C. albicans cells (strain DS1) were incubated for 1.5 h at 37 °C in the presence or absence of the indicated concentrations of Hst 5. Cells were then plated on agar plates to assess viability or supernatants were used for extracellular ATP measurements as described under "Experimental Procedures." ATP efflux is expressed in RLU and results are mean ± S.D. from duplicates from three independent experiments. Loss of cell viability is expressed as (1 - (colony forming units treated cells/colony forming units control) × 100 and data points are mean ± S.D. from 10 independent experiments.

Since the pharmacological agents provided protection to cells from Hst 5 killing, we tested whether they could prevent Hst 5-induced release of cellular ATP. C. albicans cells incubated with 10 µM CCCP prior to Hst treatment exhibited about 30% decrease in killing and Hst 5-induced ATP release was reduced by 20% (Table II). 10 mM DNP that inhibited cell killing by 69%, decreased Hst 5-induced extracellular ATP accumulation by 89%. The protective effect of azide against the release of cellular ATP by Hst 5 was even more striking, with 97% decrease in amount of extracellular ATP relative to Hst 5-treated cells that corresponded to 82% reduction in killing incidence. Together, our results demonstrate that exposure of C. albicans to Hst 5 produced release of cellular ATP and pharmacological agents which protected cells from killing inhibited ATP release, thus implying a correlation between ATP efflux and killing.

                              
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Table II
Correlation between ATP efflux and killing of C. albicans in response to Hst 5 
C. albicans cells (strain DS1) were incubated for 2 h at 37 °C with the indicated concentrations of CCCP, DNP, or azide, and left untreated or treated with Hst 5 for 1.5 h. Cells were then plated on agar plates to assess viability or supernatants were used to measure extracellular ATP as described under "Experimental Procedures." ATP efflux is expressed as percentage of ATP released from Hst 5-treated cells (100% average was 3 ± 1.5 × 106 RLU). Loss of viability is expressed as (1 - (colony forming units) treated cells/control forming units) control 1 × 100. Results are mean ± S.D. from duplicates from four independent experiments.

Effects of the Pharmacological Agents and Hst 5 on C. albicans Membrane Potential-- We next examined whether the protective effects of CCCP, DNP, and azide against Hst 5-induced ATP release and killing of C. albicans were due to dissipation of electrochemical gradient across cell membranes. Membrane potential changes were measured by flow cytometry using the fluorescent membrane potential sensitive dye DiOC5(3). DiOC5(3) is a cationic, membrane permeable dye that accumulates on polarized membranes and fluoresces intracellularly; however, upon membrane depolarization the dye uptake and fluorescence is decreased. DiOC5(3) has been previously used to measure amphotericin B-induced C. albicans membrane depolarization (33, 34).

For the flow cytometry experiments C. albicans cells were treated with CCCP, DNP, or azide at concentrations that provided maximal protection to the cells against Hst 5-induced ATP release and killing and then incubated with DiOC5(3). Preincubation of C. albicans for 10 min with 500 µM CCCP produced membrane depolarization of the entire cell population and cells remained depolarized in the presence of CCCP for 3.5 h, as evident by the 2 log units decrease in DiOC5(3) fluorescence intensity compared with control cells (Fig. 6). Similarly, 10 mM DNP reduced the fluorescence of the dye in 10 min and cell membranes remained depolarized in the presence of DNP for 3.5 h. In contrast, 10 mM azide which protected 82% of C. albicans cells from Hst 5 killing did not cause reduction in fluorescence over a period of 3.5 h (Fig. 6).


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  		Fig. 6.   Effects of CCCP, DNP, azide, and Hst 5 on C. albicans cell membrane potential. C. albicans cells (strain DS1) were incubated for 3.5 h at 37 °C with CCCP (500 µM), DNP or azide (10 mM), or cells were treated with 61 µM Hst 5 for 1.5 h. DiOC5(3) was then added for 10 min and the fluorescence distribution measured by FACSCAN flow cytometer as described under "Experimental Procedures." Data are from a representative experiment and recorded as histograms of fluorescence (FL1) versus counted cells.

The proton ionophores CCCP and DNP dissipate proton gradients, uncouple respiratory chain phosphorylation, and induce endogenous fermentation in yeast. Azide at concentrations higher than 1 mM has also been reported to function as an uncoupler in yeast, rather than a respiratory inhibitor, based on its ability to lower the intracellular pH, partially dissipate the proton gradient, and induce ethanol production (35-37, 40). However in our assays, azide differed from CCCP and DNP in that it did not affect C. albicans membrane potential. The abilities of CCCP and DNP to dissipate proton gradient across cell membranes may be related to their protective effects against killing. However, cell membranes were depolarized by DNP and CCCP in 10 min of exposure, a time when no protection against Hst 5 killing was observed (Fig. 2). Altogether, these findings suggest that electrochemical gradient, if involved, may not be solely responsible for optimal Hst 5 candidacidal activity.

Hst 5, itself, was tested for ability to cause membrane damage. C. albicans cells were treated with Hst 5, incubated with DiOC5(3), and fluorescence distribution recorded. DiOC5(3) fluorescence intensity did not decrease after 1.5 h of Hst 5 treatment, indicating that C. albicans cell membranes remained polarized (Fig. 6). The fact that ATP release from C. albicans was detected in 10 min of exposure to Hst 5 and the cell membranes remained polarized after 1.5 h of Hst 5 treatment, supports a non-lytic mechanism of exit of ATP from cells.

Effects of Hst 5 on Cellular Respiration-- To further confirm that ATP was released from intact cells, we measured C. albicans endogenous respiration (in the absence of exogenous substrate) after exposure to Hst 5. Cells treated with Hst 5 for 10 min (ATP release) or 1.5 h (inability to form colonies) were metabolically active with rates of oxygen consumption very similar to the untreated cells (Table III). In contrast, C. albicans respiration was completely blocked by exposure of the cells to antimycin A, an inhibitor of the classical respiratory chain (Table III). Thus, oxygen consumption experiments provided additional confirmation for the non-lytic release of cellular ATP in response to Hst 5, since 1.5 h after ATP release cells were actively respiring.

                              
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Table III
Effect of Hst 5 on C. albicans respiration
C. albicans cells (strain DS1) were incubated for the indicated periods at 37 °C in the presence or absence of 31 µM Hst 5 or antimycin A (1 µg/ml) and oxygen consumption was measured for 10 min as described under "Experimental Procedures." Each data point is mean ± S.D. from three independent experiments. Oxygen uptake is expressed as nanomoles of O2/min/106 cells.

Effect of Purinergic Agonists and Antagonists on Hst 5-induced Killing of C. albicans-- Channel-like pathways of ATP release in the absence of cytolysis have been described for various cell systems, including yeast (28, 41). Members of ATP-binding cassette (ABC) family of transport proteins have been implicated in conductive transport of ATP across the plasma membrane (28, 42). Furthermore, recent experimental evidence suggested that released ATP can function outside of the cell via selectively activating membrane P2 purinergic receptors to cause changes in membrane permeability (29) and even cell death (30). Therefore, we next tested whether extracellular ATP, released from the cells in response to Hst 5, may play a further physiological role in killing via activating putative ATP receptors on C. albicans. Incubation of C. albicans cells for 3.5 h with 100 µM of the purinergic agonists, the ATP analogs BzATP or ATPgamma S (30), resulted in 72 and 57% loss of cell viability, respectively (Table IV). Moreover, treatment of the cells for 2 h with purinergic antagonists suramin (100 µM) and PPAD (500 µM) (30) prior to addition of 31 µM Hst 5 prevented over 93 and 82% of Hst 5 cell killing, respectively (Table IV). These initial results provide evidence for the existence of purinergic-like receptors on C. albicans and further suggest that ATP released from cells by Hst 5 may function as a ligand for such receptors to induce cell death.

                              
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Table IV
Effect of purinergic agonists and antagonists on C. albicans killing
C. albicans cells (strain DS1) were incubated for 2 h at 37 °C with 100 µM suramin or 500 µM PPAD, followed where indicated by the addition of 31 µM Hst 5 for 1.5 h; or cells were treated with 100 µM BzATP or ATPgamma S alone for 2 h. Loss of viability was assayed as described in Table II. Each data point is the mean ± S.D. of duplicate determination from three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This is the first study to describe ATP efflux from C. albicans in response to Hst 5 that occurs in the absence of cytolysis and correlates with cell death. We report here that Hst 5-induced killing of C. albicans is initiated by a release of intracellular ATP while yeast cells are metabolically active and have polarized membranes. Furthermore, we provide evidence for the presence of putative purinergic-like receptors on these cells. Extracellular ATP, released from C. albicans by Hst 5, may activate membrane ATP receptors to cause cell death.

We began the characterization of the mechanism of Hst 5 candidacidal activity by testing pharmacological agents for effects on Hst 5-induced killing of C. albicans. Of many agents tested, the proton ionophore CCCP partially inhibited Hst 5-induced killing of C. albicans, while not affecting the viability of control cells. The effect of CCCP was specific to the cells and was not due to interaction and/or inactivation of Hst 5, since increased survival was observed when cells were first preincubated with CCCP, then washed free of drug before exposure to Hst 5 (data not shown). Uncouplers like CCCP dissipate the electrochemical gradient (Fig. 6) resulting in a decline in proton-coupled ATP synthesis, acidification of cytoplasm, inhibition of active transport of nutrients, and export of toxic drugs (35, 39, 40). Therefore, the observed inhibition of Hst 5 cell killing by CCCP can be due to the inhibition of Hst 5 initial interaction with the cell, e.g. binding or transport, or to the inhibition of an essential step in the intracellular cascade leading to cell death. The proton ionophore DNP and azide at concentrations higher than 1 mM have also been reported to function as uncouplers in C. albicans (37) and both agents protected these cells from Hst 5 killing (Fig. 3, Table I). Interestingly, the same three agents (CCCP, DNP, and azide) were shown to inhibit human neutrophil defensin-1 killing of C. albicans (43). The abilities of these drugs to reduce human neutrophil defensin-1 cytotoxic action were tentatively ascribed to their effects on mitochondrial metabolism. However, our ATP measurements revealed that ATP content in the presence of CCCP and azide was the same as in the control cells and DNP caused only a 2-fold reduction of the intracellular ATP level (Fig. 4). Therefore, the protective effects of these agents to C. albicans against Hst 5 killing cannot be explained by depletion of cellular ATP.

A striking finding of this study is that Hst 5 itself induced a drastic reduction of intracellular ATP (Fig. 4), which coincided with the 180-fold increase of the extracellular ATP (Fig. 5). ATP release occurred in 10 min (the earliest time investigated) following exposure to Hst 5 and the extracellular ATP level remained unchanged or slightly increased for up to 1.5 h. Most importantly, studies using inhibitors of Hst 5 activity suggested a relationship between ATP release and killing of C. albicans. Preincubation of the cells with concentrations of CCCP, DNP, and azide that inhibited Hst 5-induced killing by 50, 69, and 82%, also inhibited Hst 5-induced ATP release by 87, 69, and 97%, respectively (Table II). Although the exact mechanism by which CCCP, DNP, and azide induce protection is currently unclear, the use of these agents was crucial in establishing a correlation between ATP release and cell killing.

Several observations indicate that Hst 5-induced ATP release from C. albicans represents an efflux from structurally intact and metabolically active cells. First, flow cytometry experiments, using membrane potential sensitive dye showed that C. albicans cell membranes remained polarized after 1.5 h of exposure to Hst 5 (Fig. 6). Second, released ATP was not hydrolysed extracellularly for approximately 80 min (from 10 min to 1.5 h incubation of the cells with Hst 5) at 37 °C, suggesting that the cell membrane was intact and no intracellular ATPases were present to rapidly degrade ATP. This is consistent with the finding that C. albicans cells were not stained by methylene blue and acridine orange after 1 h of exposure to Hst 1 (13). Finally, C. albicans cells treated with Hst 5 for either 10 min (ATP release) or 1.5 h (complete cell killing measured by inability to form colonies) were consuming oxygen at rates similar to untreated cells, indicating active respiration (Table III). Although the concentration of ATP in the cells is an indicator of viability and ATP is rapidly degraded when cells die, actively metabolizing organisms with inhibited DNA replication have also been described (44). Perhaps, the loss of cellular ATP, caused by Hst 5, forces the cell to maintain the proton gradient across the membranes at the expense of all of the newly synthesized ATP. Whether cells maintain their integrity, but have no energy to replicate, or Hst 5-induced ATP release initiates processes leading to later loss of cell structure will require further study. However, the effect of Hst 5 on cell viability, at least during the 1.5 h of treatment, is not a result of a destructive action.

The findings described here suggest a specific cellular mechanism for exit of ATP in response to Hst 5. The known mechanisms of specific ATP release include: 1) facilitated transport of ATP through a transporter down a favorable concentration gradient; 2) conductive transport through an ATP-specific or anion channel; and 3) release of ATP by exocytosis (28). Although little is known about ATP-specific channels, members of ATP-binding cassette (ABC) proteins, such as P-glycoprotein (45) and cystic fibrosis transmembrane conductance regulator (46), have been implicated in conductive transport of ATP. ABC transporters which carry out transport of a wide range of substrates or function as efflux pumps have been identified in Saccharomyces cerevisiae and resistance to azole antifungal agents in C. albicans has been shown to be mediated by the ABC transporters CDR1 and CDR2 (47, 48). Furthermore, S. cerevisiae released ATP in response to the toxic H+/K+ ionophore nigericin, while the cell membrane permeability was not altered (41) and a plasma membrane ATP-specific transporter was purified from Aspergillus niger that was activated by the lipid-reactive antibiotic mycobacillin (49).

Extracellular ATP can cause striking changes in membrane permeability through activating membrane receptors for extracellular nucleotides (purinoceptors, P2) expressed on susceptible cells (50). Among the P2 receptors, P2Y are G-protein-coupled receptors and P2X are ligand-gated channels more selectively localized on excitable cells (31, 51). By contrast, P2Z (P2X7) receptors operate as ATP-gated pores and cells expressing these receptors are killed when stimulated with agonists, ATP, or ATP analogues (30, 31, 52). ATP binding, but not hydrolysis is needed for receptor activation. We showed here that stimulation of C. albicans cells with the purinergic agonists BzATP or ATPgamma S resulted in 72 and 57% loss of cell viability, respectively; and purinergic antagonists suramin and PPAD prevented Hst 5 killing (Table IV), thus, providing evidence for the presence of Candidal purinergic-like receptors. The P2Z/P2X7 receptor is selectively activated by ATP in its fully dissociated tetra-anionic form (ATP4-) and extracellular Ca2+ and Mg2+ prevent channel opening and cell lysis (30, 31, 53). In this context, it is of interest to note that Ca2+ and Mg2+ have been reported to abolish Hst 5 killing C. albicans (11).

Activation of P2Z/P2X7 receptors requires relatively high doses of extracellular ATP, from 100 µM to 1 mM. The most likely source of this amount of ATP is release from stressed or injured cells, intracellular concentration of ATP is 5-10 mM for higher eukaryotic cells (31) and about 1 mM for yeast cells (35). Our data showed that Hst 5 induced release of more than half of the reported C. albicans intracellular ATP, a sufficient amount to activate P2Z/P2X7 receptors. ATP-pulsed cells expressing these receptors appear to initially maintain their integrity and physiological function, yet cytolysis occurs 10 to 12 h after receptor stimulation (31, 54). Similarly, C. albicans cells were actively respiring and had polarized membranes after 1.5 h treatment with Hst 5, but could not subsequently form colonies after 24 h. Admittedly, purinergic-like receptors have not been described on yeast and, P2Z/P2X7 cytotoxic receptors have been found mainly on immune and inflammatory cells. However, C. albicans is known to possess complement receptors, CR2- and CR3-like proteins for C3d and iC3b, that are mostly expressed on the B lymphocytes and phagocytic cells (55, 56).

Our findings provide support for the concept that extracellular ATP plays a role in cell killing and raise the possibility of a novel mechanism for Hst 5 cytotoxic action that include: (a) killing of C. albicans is initiated with release of cellular ATP; (b) ATP release is via non-lytic, perhaps, conductive pathway; (c) extracellular ATP, released in response to Hst 5, may activate putative purinergic receptors on C. albicans to ultimately induce cell death.

Characterization of many other important aspects of Hst 5 cytotoxic action will require further study. It is currently unknown whether Hst 5 induces efflux of cellular ATP through binding to a cell surface component or needs to be transported into the cells. Furthermore, it is yet unanswered how ATP is released from the cells and whether ATP-binding cassette proteins are involved in conductive transport of ATP. We have previously reported the presence of C. albicans binding sites for salivary Hsts and a 67-kDa yeast Hst 5-binding protein (17). This protein is of similar size to the purified plasma membrane ATP transporter from A. niger (49) and may function as an ATP channel activated upon Hst 5 binding. Purification and identification of the Hst 5-binding protein would be an important step in understanding the killing mechanism. Exploring the protective effects of the uncouplers to C. albicans cells against Hst 5 killing will aid in understanding the biochemical mechanism of Hst 5 candidacidal activity. Finally, the molecular identity of ATP receptors involved in Hst 5 killing of C. albicans is crucial in understanding the cytotoxic mechanism. The mechanism described here for Hst 5-induced yeast killing has not been evaluated for other antifungal agents and antimicrobial proteins. Consequently, it will be important to determine whether it represents a common antifungal mechanism or it is unique to Hst 5.

In conclusion, major disadvantages of currently used antifungal drugs to treat candidiasis are their toxicity and the development of resistant yeast strains. In contrast, salivary Hsts are nontoxic to humans and yet potent candidacidal agents even with drug-resistant strains. While 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.

    ACKNOWLEDGEMENTS

We thank Dr. Michel J. Levine and Dr. Jeremy Bruenn for helpful discussions, Drs. Philip Loverde and Arvind Thakur for the use of the luminometer and advice on ATP assays, and Dr. N. Baev for interest and critical comments regarding this manuscript.

    FOOTNOTES

* This work was supported by United States Public Health Grants DE10641 and DE12159 from the National Institute of Dental and Craniofacial Research, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 310 Foster Hall, SUNY at Buffalo Main St. Campus, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-3067; Fax: 716-829-3942; E-mail: Mira_Edgerton{at}sdm.buffalo.edu.

    ABBREVIATIONS

The abbreviations used are: Hsts, histatins; Hst 5, histatin 5; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DNP, dinitrophenol; BzATP, 3'-O-(4-benzoylbenzoyl)-ATP; PPAD, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid; DiOC5(3), 3,3'-dipentyloxacarbocyanine iodide; RLU, relative light units; ATPgamma S, adenosine 5'-O-(thiotriphosphate).

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RESULTS
DISCUSSION
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