The Quorum-Sensing Molecule Farnesol Is a Modulator of Drug Efflux Mediated by ABC Multidrug Transporters and Synergizes with Drugs in Candida albicans^,

Abstract

Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent K_m without affecting the V_max values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC₈₀ values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB).

INTRODUCTION

Infections caused by the dimorphic opportunistic pathogen Candida albicans are treated by antifungal agents. Widespread and prolonged usage of antifungals, in recent years, has led to the emergence of strains of Candida which display multidrug resistance (MDR) (15, 16, 19). Among the various mechanisms used by the fungus to gain resistance toward antifungal therapy, enhanced drug export represents an important strategy. Most of the clinically drug-resistant isolates of C. albicans are shown to overexpress genes encoding CaCDR1, CaCDR2, or CaMDR1 drug efflux pump proteins. CaCDR1 and CaCDR2 belong to ATP binding cassette (ABC) transporters, which use energy driven from ATP hydrolysis to transport drugs outside the cells, while CaMDR1, a major facilitator superfamily (MFS) protein, utilizes proton gradient for drug extrusion (20, 23). Notably, major multidrug transporters of Candida that belong to different superfamilies of proteins are functionally identical in expelling drugs but differ mechanistically in achieving drug expulsion. Among various strategies employed to combat MDR, blocking or modulating the function of the drug efflux pump proteins represents an attractive approach (35).

MDR in cancer cells is an obstacle to effective chemotherapy. ABC transporters, including ABCB1, ABCC1, and ABCG2, play an important role in the development of frequently encountered MDR in cancer cells (29). Here again, among different approaches employed to overcome MDR, inhibition of the drug extrusion pump activity represents an attractive approach (29, 36). Many clinically relevant anticancer drugs, such as Vinca alkaloids (vinblastine and vincristine), anthracyclines (doxorubicin and daunorubicin), taxenes (paclitaxel and docetaxel), epipodophylltoxins (etoposide and teniposide), camptothecins (topotecan), and anthracenes, are identified as modulators of human ABC transporters which offer great hope in successful cancer chemotherapy (36). In comparison, modulators of MDR pump proteins in pathogenic yeasts are only beginning to be characterized. There are already examples of compounds, such as enniatins, milbemycins, synthetic d-octapeptides, isonitrile, and unnarmicins, which modulate drug efflux by inhibiting the fungal multidrug transporters (11, 35). We have earlier shown that disulfiram, an antabuse, acts as a modulator of CaCdr1p by inhibiting oligomycin-sensitive ATP hydrolysis and affecting drug binding sites in CaCdr1p (33). Recently, polyphenol curcumin (CUR) has also been shown to be a specific modulator of rhodamine 6G (R6G) efflux mediated by CaCdr1p, CaCdr2p, and ScPdr5p (27). CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by the prazosin analog [¹²⁵I]iodoarylazidoprazosin without affecting ATPase activity (27).

Farnesol (FAR), a quorum-sensing molecule (QSM), is a precursor for the synthesis of sterols in C. albicans; it also blocks the morphological transition and biofilm development in Candida (10). FAR is known to be involved in triggering apoptosis in human oral squamous carcinoma cells (24). In mammalian cells, FAR interferes with calcium signaling and membrane fluidity (24). Studies on quorum sensing suggest its involvement in fungus-bacterium interactions and biofilm formation (34). Notably, FAR also induces apoptosis in a number of fungal species (4, 25). A global protein expression profiling following FAR treatment in C. albicans revealed mitochondrial degradation, reactive oxygen species (ROS) accumulation, caspase activation, and apoptosis as a cause of cell death (30). In this study, we provide evidence that FAR could also specifically modulate drug extrusion mediated by ABC transporters, such as CaCdr1p and CaCdr2p, without affecting the MFS transporter, such as CaMdr1p. It specifically modulates the efflux of substrates, such as R6G and fluconazole (FLC), whereas it has no effect on the efflux of substrates like Nile red (NR) and methotrexate (MTX). FAR at its nonlethal concentrations also synergizes with azoles and polyenes. Together, we show that FAR is a specific modulator of the efflux of drugs mediated by ABC transporter proteins, and it also displays synergism to antifungals by accumulating ROS and resulting in an early cell death.

MATERIALS AND METHODS

Materials.

Rhodamine 6G (R6G), 2,4-dinitrophenol (DNP), 2-deoxy-d-glucose (DOG), oligomycin, 3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), Nile red (NR), and other molecular-grade chemicals were obtained from Sigma Chemicals Co. (St. Louis, MO). [³H]fluconazole ([³H]FLC; specific activity, 19 Ci/mmol) was custom synthesized from Amersham Biosciences, United Kingdom, and [³H]methotrexate ([³H]MTX; specific activity, 8.60 Ci/mmol) was procured from Amersham Biosciences, United Kingdom. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), ascorbic acid (AA), and other molecular-grade chemicals were obtained from Sigma Chemicals Co. (St. Louis, MO). The annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from BD Biosciences.

Yeast strains and growth media.

The strains used in this study are listed in Table 1. The yeast strains were cultured in yeast extract-peptone-dextrose (YEPD) broth (BIO101; Vista, CA) or RPMI 1640 medium. For agar plates, 2.5% (wt/vol) Bacto agar (Difco, BD Biosciences, NJ) was added to the medium. All strains were stored as frozen stocks with 15% glycerol at −80°C. Before each experiment, cells were freshly revived on YEPD plates from the stock.

Table 1.

Strains used in this study

Strain no.	Strain	Genotype or description	Reference
1	AD1-8u−	matα pdr1-3 his1 ura3 Δyor1::hisG Δsnq2::hisG Δpdr5::hisG Δpdr10::hisG Δpdr11::hisG Δycf1::hisG Δpdr3::hisG Δpdr15::hisG	3
2	AD-CDR1	AD1-8u− cells harboring CaCDR1-GFP ORF integrated at PDR5 locus	32
3	AD-CaMDR1	AD1-8u− cells harboring CaMDR1-GFP ORF integrated at PDR5 locus	17
4	AD-CDR2	AD1-8u− cells harboring CaCDR2-GFP ORF integrated at PDR5 locus	13
5	AD-PDR5	AD1-8u− cells harboring ScPDR5-GFP ORF integrated at PDR5 locus	13
6	SC5314	Wild-type strain of C. albicans	9
7	Gu5	Fluconazole-resistant clinical isolate	5
8	F5	Fluconazole-resistant clinical isolate	6

Efflux of rhodamine 6G.

Efflux of R6G was determined essentially using a previously described protocol (32). Briefly, approximately 1 × 10⁶ yeast cells from overnight-grown culture were transferred into YEPD medium and allowed to grow for 5 h. Cells were pelleted, washed twice with phosphate-buffered saline (PBS; without glucose), and resuspended as a 2% cell suspension, which corresponds to 10⁸ cells (wt/vol) in PBS without glucose. The cells were then deenergized for 45 min in DOG (5 mM) and DNP (5 mM) in PBS (without glucose). The deenergized cells were pelleted, washed, and then resuspended as a 2% cell suspension (wt/vol) in PBS without glucose, to which R6G was added at a final concentration of 10 μM and incubated for 40 min at 30°C. The equilibrated cells with R6G were then washed and resuspended as a 2% cell suspension (wt/vol) in PBS without glucose. Samples (1-ml volumes) were withdrawn at the indicated time and centrifuged at 9,000 × g for 2 min. The supernatant was collected, and absorption was measured at 527 nm. Energy-dependent efflux (at the indicated time shown in Fig. 1) was measured after the addition of glucose (2%) to the cells resuspended in PBS (without glucose). Glucose-free controls were included in all the experiments. For competition assays, FAR (100 μM) was added to the deenergized cells 5 min before the addition of R6G and allowed to equilibrate.

Fig. 1.

Effect of FAR on R6G transport. Extracellular R6G concentrations in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaCdr1p (AD-CDR1) (A), CaCdr2p (AD-CDR2) (B), and ScPdr5p (AD-PDR5) (C). (D) Wild-type strain SC5314 of C. albicans. The energy-dependent R6G efflux was initiated by adding glucose (2%; indicated by an arrow) and quantified by measuring the absorbance of the supernatant at 527 nm. The values are the means ± standard deviations (indicated by error bars) from three independent experiments.

Measurement of drug accumulation.

The accumulation of [³H]FLC (specific activity, 19 Ci/mmol), [³H]MTX (specific activity, 8.60 Ci/mmol), and fluorescent NR was determined essentially by the methods described previously (17, 21). Briefly, cells from mid-log phase (5 × 10⁶) were centrifuged at 3,000 × g for 3 min and resuspended in PBS as a 2% cell suspension. For accumulation studies, 100 nM FLC and 25 μM MTX were routinely used (17). FAR (100 μM) was added 5 min before the addition of drugs and allowed to equilibrate. One hundred microliters of cell suspension containing drugs or drugs with FAR were incubated at 30°C for 40 min, filtered rapidly, and washed twice with PBS (pH 7.4) on a Millipore manifold filter assembly using a 0.45-μm-pore-size cellulose nitrate filter (Millipore). The filter discs were dried and put in cocktail O, and the radioactivity was measured in a liquid scintillation counter (Beckman). The accumulation was expressed as picomoles/milligram (dry weight). Accumulation of NR was measured by flow cytometry with a FACsort flow cytometer (Becton-Dickson Immunocytometry Systems, San Jose, CA) as described previously (21).

ATPase assay.

ATPase activity of the plasma membrane fractions was measured as oligomycin-sensitive release of inorganic phosphate either alone or in the presence of FAR (100 μM) as described previously (32).

Time-kill assays.

C. albicans cells at a concentration of 10³ CFU/ml were inoculated in RPMI 1640 medium containing either FAR or antioxidant ascorbic acid (AA) alone or a combination of both FAR and AA as detailed previously (26). At predetermined time points (0, 4, 8, 12, 16, 20, and 24 h) (at 30°C incubation; agitation, 200 rpm), a 100-μl aliquot was removed, serially diluted (10-fold) in 0.9% saline, and plated on YEPD agar plates. Colony counts were determined after incubation at 30°C for 48 h (26).

Measurement of ROS production.

Endogenous amounts of ROS were measured by a fluorometric assay with 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (26). Briefly, the cells were adjusted to an optical density at 600 nm (OD₆₆₀) of 1 in 10 ml of PBS and centrifuged at 2,500 × g for 15 min. The cell pellet was then resuspended in PBS and treated with appropriately diluted AA for 1 h or was left untreated at room temperature. After incubation with FAR at 37°C for different time intervals as indicated, 10 μM DCFH-DA in PBS was added. The fluorescence intensities (excitation and emission of 485 and 540 nm, respectively) of the resuspended cells were measured with a spectrofluorometer (Varian; Cary Eclipse).

Analysis of apoptotic markers.

Protoplasts of C. albicans were stained with propidium iodide (PI) and FITC-labeled annexin V by using the annexin V-FITC apoptosis detection kit (BD Biosciences) to assess cellular integrity and the externalization of phosphatidylserine (PS) as described earlier (26). The cells were analyzed by using a fluorescence-activated cell sorter (FACS) caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) using a 488-nm excitation and a 515-nm band pass filter for FITC detection and a filter >560 nm for PI detection. A total of 10,000 events were counted at the flow rate. Data analysis was performed using Cell Quest software (Becton Dickinson Immunocytometry Systems) (26).

Liquid susceptibility assay.

The interaction of FAR with FLC, ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB) was evaluated by the checkerboard method recommended by the NCCLS and expressed as the sum of the fractional inhibitory concentration index (FICI) for each agent. The FIC of each agent is calculated as the MIC of this agent in combination divided by the MIC of this agent alone (26). In brief, serial double dilutions of the anticandidal compounds were prepared ranging from 0.25 to 128 μg/ml for FLC, 0.019 to 10 μg/ml for KTC, and 0.019 to 10 μg/ml for MCZ and AMB. After drug dilutions were made, a 100-μl suspension of Candida strains adjusted to 5 × 10⁵ CFU/ml was added to each well and cultured at 30^οC for 48 h in RPMI 1640 medium. Then visual reading of MICs was performed, and OD₆₀₀ values were measured. The background OD value was subtracted from the OD value of each well. Each checkerboard test generates many different combinations, and by convention the FIC value of the most effective combination is used in calculating the FIC index. FICI was calculated by adding both FICs: FICI = FICA + FICB = C_A^comb/MIC_A^alone + C_B^comb/MIC_B^alone, where MIC_A^alone and MIC_B^alone are the MICs of drug A and B when acting alone and C_A^comb and C_B^comb are concentrations of drugs A and B at the isoeffective combinations, respectively. Off-scale MICs were converted to the next highest or next lowest doubling concentration. The FICI was interpreted as synergistic when it was ≤0.5, as antagonistic when >4.0, and as indifferent at any value in between (26).

MTT assay.

The cytotoxic effect of FAR was determined by MTT assay (1, 2, 30). Yeast cells (10⁴) were seeded into 96-well plates in the absence and in the presence of various concentrations of FAR (25 to 500 μM) and grown for 48 h at 30°C. MTT solution (100 μl) was added to each well and incubated for 3 to 4 h, and 200 μl of isopropanol was added to stop the reaction. The absorbance of the whole microtiter plate was measured using a microplate spectrophotometer at 570 nm with a reference wavelength of 650 nm. Cell survival (as a percentage of the control) equals the mean absorbance in the test well divided by the mean absorbance in control wells, multiplied by 100.

RESULTS

FAR inhibits R6G efflux mediated by ABC transporters.

In this study, we explored the modulator effect of FAR on MDR efflux proteins of C. albicans. For this, we monitored the transport of well-known efflux substrate R6G (10 μM) in the presence of FAR (100 μM) in cells where green fluorescent protein (GFP)-tagged ABC transporters, such as CaCdr1p (CDR1-GFP), CaCdr2p (CDR2-GFP), and ScPdr5p (PDR5-GFP), were stably overexpressed from a genomic PDR5 locus in the Saccharomyces cerevisiae AD1-8u⁻ mutant, which is deleted in seven ABC transporters (15). We have shown earlier that overexpression of GFP-tagged ABC proteins of C. albicans offered expression levels sufficient for the biochemical characterization of the transporters (17, 32). It should be mentioned that the functionality of the GFP-tagged version of MDR transporters remains similar to their respective untagged proteins (13, 17, 32). As depicted in Fig. 1, there was no efflux of R6G in control energized AD1-8u⁻ cells. In contrast, the AD1-8u⁻ cells overexpressing CDR1-GFP showed time- and energy-dependent efflux of R6G. This was evident from a steady increase in the extracellular concentration of R6G (Fig. 1A). We further evaluated if the observed inhibitory effects of FAR on R6G transport could be extended to CaCdr1p homologues, such as CaCdr2p and ScPdr5p, which were also expressed in similar heterologous backgrounds (15). It was observed that FAR could inhibit the efflux of R6G mediated by both the proteins (Fig. 1B and C). Of note, FAR could also modulate R6G efflux in C. albicans wild-type (WT) (SC5314) cells (Fig. 1D); however, for subsequent detailed studies, we used the heterologous host AD1-8u⁻ strain overexpressing MDR transporters.

FAR is a substrate-specific modulator of ABC proteins.

While FAR was able to modulate R6G efflux mediated by all the tested ABC transporter proteins, such as CaCdr1p, CaCdr2p, and ScPdr5p (Fig. 1A, B, and C), we explored if FAR could also affect other known substrates of ABC transporters, such as FLC, MTX, and NR. As depicted in Fig. 2A, there was no change in the accumulation of FLC in AD1-8u⁻ control host cells, while cells overexpressing CaCdr1p showed reduced accumulation of FLC (enhanced efflux). Interestingly, the presence of FAR (100 μM) blocked the transport of FLC (∼80%) mediated by CaCdr1p-expressing cells, which was evident by its increased accumulation (decreased efflux). As depicted in Fig. 2B and C, the S. cerevisiae cells overexpressing CaCdr1p showed decreased accumulation of MTX and NR (increased efflux), respectively; however, unlike in the case of FLC, the presence of FAR (100 μM) had no effect on MTX or NR transport.

Fig. 2.

FLC, MTX, and NR transport in CaCdr1p-overexpressing S. cerevisiae cells. (A) [³H]FLC accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaCdr1p (AD-CDR1). Cells were incubated with either [³H]FLC (100 nM; specific activity, 19 Ci/mmol) or [³H]FLC and FAR (100 μM). The accumulated [³H]FLC was measured 40 min after the addition of glucose (2%). The values indicated by the bars represent the means ± standard deviations (indicated by error bars) from three independent experiments. (B) [³H]MTX accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaCdr1p (AD-CDR1). Cells were incubated with either [³H]MTX (25 μM; specific activity, 8.60 Ci/mmol) or [³H]MTX and FAR (100 μM). The accumulated [³H]MTX was measured 40 min after the initiation of efflux, using a liquid scintillation counter (Beckman). The values indicated by the bars represent the means ± standard deviations (indicated by error bars) from three independent experiments. (C) NR accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaCdr1p (AD-CDR1). Cells were incubated with either NR (3.5 μM) or NR and FAR (100 μM). The accumulated NR was measured 40 min after the initiation of efflux.

FAR has no effect on the transport mediated by MFS protein.

We explored if FAR could also affect a multidrug transporter belonging to the MFS superfamily, and we examined the efflux mediated by CaMdr1p (an MFS transporter) expressed in similar heterologous backgrounds. For this, we monitored the transport of well-known substrates of CaMdr1p, such as FLC, MTX, or NR. As shown in Fig. 3, FAR could not block the efflux of FLC (Fig. 3A), MTX (Fig. 3B), and NR (Fig. 3C) mediated by CaMdr1p overexpressed in similar backgrounds.

Fig. 3.

FLC, MTX, and NR transport in CaMdr1p-overexpressing S. cerevisiae cells. (A) [³H]FLC accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaMdr1p (AD-CaMDR1). Cells were incubated with either [³H]FLC (100 nM; specific activity, 19 Ci/mmol) or [³H]FLC and FAR (100 μM). The accumulated [³H]FLC was measured 40 min after the addition of glucose (2%). The values indicated by the bars represent the means ± standard deviations (indicated by error bars) from three independent experiments. (B) [³H]MTX accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaMdr1p (AD-CaMDR1). Cells were incubated with either [³H]MTX (25 μM; specific activity, 8.60 Ci/mmol) or [³H]MTX and FAR (100 μM). The accumulated [³H]MTX was measured 40 min after the initiation of efflux, using a liquid scintillation counter (Beckman). The values indicated by the bars represent the means ± standard deviations (indicated by error bars) from three independent experiments. (C) NR accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaMdr1p (AD-CaMDR1). Cells were incubated with either NR (3.5 μM) or NR and FAR (100 μM). The accumulated NR was measured 40 min after the initiation of efflux.

FAR is not a substrate of MDR pumps.

The fact that FAR inhibits drug transport suggests that FAR could be acting as a competing substrate for the MDR transporters. Therefore, cytotoxicity of FAR in the control (AD1-8u⁻) and ABC/MFS transporter-expressing cells (AD-CDR1, AD-CDR2, AD-PDR5, AD-CaMDR1) was determined by using MTT assay. As shown in Fig. 4, the 50% inhibitory concentrations (IC₅₀s) of FAR for control host strain AD1-8u⁻, AD-CDR1, AD-CDR2, AD-PDR5, and AD-CaMDR1 were 432.44 ± 3.132, 377.99 ± 3.090, 415.66 ± 2.957, 402.87 ± 4.583, and 421.14 ± 3.355 μM, respectively. The relative resistance factor was between ∼0.874 and 1.0 (Fig. 4), indicating that the overexpression of different pump proteins could not affect IC₅₀s of FAR. This implies that FAR could not be transported by both the control cells and the efflux pump protein-overexpressing cells. As depicted in Fig. 4, the IC₅₀s for control (AD1-8u⁻) cells and cells expressing the transporters (ABC or MFS) were not very different up to 400 μM FAR, ensuring that at the tested concentration of FAR, the viability of cells was not affected.

Fig. 4.

Effect of FAR on the viability of S. cerevisiae cells overexpressing MDR pumps as determined by MTT assay. (A) Percent cell survival in control cells (AD1-8u⁻) and in cells overexpressing ABC/MFS transporters. The experiments were conducted in triplicates, and the values represent means ± standard deviations from three independent experiments. The table depicts the IC₅₀s and the relative resistance factors for AD1-8u⁻, AD-CDR1, AD-CDR2, AD-PDR5, and AD-CaMDR1 in the presence of FAR.

FAR competitively inhibits R6G efflux.

The efflux of R6G was inhibited by FAR in a concentration-dependent manner, with an IC₅₀ of 30 ± 5 μM (Fig. 5A). The Lineweaver-Burk plot analysis revealed that FAR competitively inhibited R6G efflux with an increase in apparent K_m (6.39 to 17.68 μM) and with no change in the V_max values (Fig. 5B). Notably, FAR at the modulator concentration did not affect the ATPase activity of CaCdr1p (Fig. 5C) and had no effect on the leakage of R6G (Fig. 1A).

Fig. 5.

Biochemical analysis of CaCdr1p in the presence of FAR. (A) For the competition assay of R6G and FAR, CaCdr1p-overexpressing S. cerevisiae cells were incubated with either R6G (10 μM) or R6G (10 μM) and FAR (10 to 100 μM). R6G efflux was monitored 40 min after the addition of glucose (2%). (B) Lineweaver-Burk plot of CaCdr1p-mediated R6G efflux in the presence of FAR 5 min after the addition of glucose (2%). The x axis (1/S) represents the various concentrations (μM) of R6G used, and the y axis (1/V) shows the rate of release of R6G in the absence (0X) and in the presence of 50 μM (5X) and 100 μM (10X) of FAR. The rate of each reaction was calculated as nanomoles of R6G released/minute/5 × 10⁶ cells. (C) Effect of FAR on the ATPase activity of CaCdr1p. Plasma membranes from cells overexpressing CaCdr1p were incubated with and without 100 μM FAR in the ATPase buffer. The assay was performed essentially as described in Materials and Methods.

FAR is synergistic to tested drugs.

FAR is known to show antifungal activity against various species of Candida (30). Here, we confirm that FAR elicited antifungal activity against the WT strain and azole-resistant clinical isolates of Candida. In addition, we also observed that FAR displays synergistic interactions with the known antifungals. A positive interaction with the drugs as demonstrated by checkerboard assays was observed. For example, in the WT strain of C. albicans, the MIC₈₀ values of FLC, KTC, MCZ, and AMB alone were 0.5, 0.5, 0.2, and 0.31 μg/ml, respectively, which in combination with FAR was reduced to 0.00775, 0.062, 0.025, and 0.234 μg/ml, resulting in 64-, 8-, 8-, and 16-fold drops in the MIC₈₀ values, respectively. A FICI value of less than 0.5 suggested synergism between the tested drugs and FAR (Table 2). The time-kill curves in the presence of the drugs at synergistic concentrations either alone or in combination confirmed the checkerboard results (Fig. 6).

Table 2.

Checkerboard assay of FLC, KTC, MCZ, AMB, and FAR against the WT strain of C. albicans in the absence and presence of AA

Antifungal agent	MIC80 of each agent (μg/ml)			FICI	FICI with AA
	Alone	Combination	Combination with AA
FLC	0.500	0.007	0.062	0.140	0.374
FAR	88.00	11.00	22.00
KTC	0.062	0.500	0.500	0.249	0.500
FAR	88.0	11.00	22.00
MCZ	0.062	0.015	0.015	0.366	0.366
FAR	88.00	11.00	11.00
AMB	0.310	0.019	0.078	0.125	0.500
FAR	88.00	5.500	22.00

Fig. 6.

Time-kill curves in the presence of drugs and FAR in the WT strain SC5314. FAR in combination with (i) FLC or FLC and AA, (ii) KTC or KTC and AA, (iii) MCZ or MCZ and AA, and (iv) AMB or AMB and AA. The concentrations used are FLC (0.00775 μg/ml) and FAR (11 μg/ml), KTC (0.062 μg/ml) and FAR (5.5 μg/ml), MCZ (0.025 μg/ml) and FAR (22 μg/ml), and AMB (0.078 μg/ml) and FAR (11 μg/ml). Ascorbic acid (AA) is used at a concentration of 25 mM. CNT, control having no drug.

FAR at nontoxic concentrations display synergy with drugs in azole-resistant isolates.

The synergistic effect of FAR in combination with FLC/KTC/MCZ/AMB could be extended against azole-resistant isolates of C. albicans, viz., Gu5 and F5. The Gu5 and F5 isolates represent AR strains derived from azole-sensitive isolates Gu4 and F2, respectively (5, 6). Both Gu5 and F5 strains display high MIC₈₀ values, which is predominantly due to an overexpression of CaCDR1 and CaMDR1, respectively (5, 6). FAR showed synergistic interactions with the drugs in clinical isolates Gu5 and F5, as demonstrated by FICI values of less than 0.5 (Table 3). The considerable drop in the MIC₈₀ values of the drugs (1.5- to 16-fold) in the clinical isolates points to the efficacy of FAR even in azole-resistant clinical isolates. Further, these interactions were confirmed by the time-kill curves (see Fig. S1 and S2 in the supplemental material).

Table 3.

Checkerboard assay of FLC, KTC, MCZ, AMB, and FAR against azole-resistant isolates Gu5 and F5

Clinical isolate and antifungal agent	MIC80 of each agent (μg/ml)		FIC	FICI
	Alone	Combination
Gu5
FLC	128	8	0.06	0.310
FAR	88	22	0.25
KTC	128	16	0.125	0.375
FAR	88	22	0.25
MCZ	128	16	0.125	0.250
FAR	88	11	0.125
AMB	0.31	0.0195	0.0629	0.125
FAR	88	5.5	0.0625
F5
FLC	128	8	0.06	0.185
FAR	88	11	0.125
KTC	64	8	0.125	0.250
FAR	88	11	0.125
MCZ	64	16	0.25	0.375
FAR	88	11	0.125
AMB	0.31	0.0195	0.0629	0.125
FAR	88	5.5	0.0625

The synergism between FAR and drugs raises ROS levels.

As depicted in Fig. 7A, the treatment of drugs such as FLC/KTC/MCZ/AMB or FAR alone at nontoxic, synergistic concentrations did not influence the levels of endogenous ROS; however, in combination with the drugs, FAR considerably augmented ROS levels. Since the combination of drugs and FAR generated ROS, we speculated whether reversal of it could affect the synergism. For this, we performed checkerboard assays in the presence of an antioxidant ascorbic acid (AA) and could indeed observe a partial reversal of synergism with a simultaneous decrease in ROS levels (Table 2 and Fig. 7A).

Fig. 7.

ROS levels in the presence of FAR/drugs and detection of apoptosis. (A) Amounts of ROS produced in WT strain SC5314 following treatment with either drugs alone or in combination with FAR or FAR and AA. The drugs (concentrations) used are FLC (0.00775 μg/ml) and FAR (11 μg/ml), KTC (0.062 μg/ml) and FAR (5.5 μg/ml), MCZ (0.025 μg/ml) and FAR (22 μg/ml), and AMB (0.078 μg/ml) and FAR (11 μg/ml). AA is used at a concentration of 25 mM. FAR-F, FAR-K, FAR-M, and FAR-A show ROS generated due to FAR alone when used at a concentration of 11 μg/ml, 5.5 μg/ml, 22 μg/ml, and 11 μg/ml, respectively. (B) Detection of apoptosis after costaining of cells with annexin V and PI in control untreated WT strain SC5314 and in cells following treatment with either FLC or AMB either alone or in combination with FAR or FAR and AA. The drugs (concentrations) used are FLC (0.00775 μg/ml), FAR (11 μg/ml), AMB (0.078 μg/ml), and AA (25 mM). The concentrations used are chosen from the checkerboard data.

Increased ROS leads to apoptosis.

FAR produced by C. albicans has the ability to induce apoptosis via ROS generation and upregulation of a metacaspase, MCA1, that is involved in apoptosis or programmed cell death (PCD) (30). In addition, our previous studies of drug-drug interactions have shown that raised ROS levels due to synergy between drugs and polyphenol CUR lead to apoptosis (26). We checked if the augmented ROS due to the combination of FAR and tested drugs triggers apoptosis. To explore this, we selected two representative drugs, viz., FLC and AMB, for further analysis. As shown in Fig. 7B, there was almost no apoptotic population (∼1.5 to 2%) in cells treated with nontoxic synergistic concentrations of FAR, FLC, or AMB alone. However, there was a significant population of apoptotic cells when FAR was used in combination with FLC (18.44%) or AMB (10.44%). The treatment of cells with AA could partly reverse the percentage of apoptotic population (4.22% and 3.15% in FLC-FAR and FLC-AMB combinations, respectively).

DISCUSSION

Among the various transporter genes identified in the C. albicans genome (7, 8), there are overwhelming clinical and experimental evidences to suggest that out of 28 ABC transporters, only two of its members, CaCdr1p and CaCdr2p, and only CaMdr1p out of a total 95 of members of MFS transporters are major determinants of MDR (18, 23). Therefore, the search for novel and potent modulators which can block and reverse the drug extrusion mediated by these efflux proteins represents an attractive strategy of anti-Candida therapy. While there are several compounds which are shown to reverse MDR mediated by Pgp (human homologue of CaCdr1p and CaCdr2p) and are at various stages of clinical trials, there are not many instances of compounds which could inhibit/reverse drug extrusion mediated by fungal multidrug transporters (12, 14, 22, 31). We have been exploring modulators/inhibitors of MDR pump proteins which could block the efflux of drugs from fungal cells. For example, we have earlier reported that purified polyphenol CUR and disulfiram, a drug used in treating alcoholism, act as modulators of CaCdr1p and thus could reverse drug efflux from Candida cells (27, 33). However, the mechanisms by which these two modulators affect the functioning of the drug efflux pump seem to differ. For example, while CUR competes for the drug binding sites without affecting the ATPase activity (27), disulfiram blocks the drug efflux by competing with the substrate binding sites as well as with a simultaneous abrogation of ATPase activity of CaCdr1p (33).

In the present study, we provide first evidence that a quorum-sensing, antifungal FAR is a modulator of drug efflux pump proteins of Candida. Thus, FAR could reverse the extrusion of specific compounds mediated exclusively by the ABC drug transporters, such as CaCdr1p, CaCdr2p, and ScPdr5p, since the efflux mediated by an MFS transporter CaMdr1p could not be inhibited by FAR. This selectivity of modulators to one type of superfamily of transporters is not uncommon (27). We had earlier observed that the modulator CUR could affect the efflux activity mediated only by the ABC drug transporters (27). If one considers the physiological concentration of FAR, which ranges between 30 and 40 μM (10), and compares it with the IC₅₀ of the drug efflux modulation concentration (30 μM), it implies that in vivo FAR, in addition to being a QSM, could also modulate transport.

We show that FAR is not a transport substrate of ABC transporter CaCdr1p but that it can specifically modulate efflux of R6G and FLC mediated by it. This is not surprising, since it has been observed that several modulators could inhibit drug transport without being a substrate of efflux pump proteins (27, 33). Our relative resistance factor values from the cytotoxicity data (Fig. 4) reinforce the fact that the presence or absence of efflux pump proteins did not confer additional advantage toward resistance against FAR or affected the growth and viability of transporter-expressing cells. Our data that FAR is not a transport substrate but that it still competitively modulates R6G transport without affecting the ATPase activity in CaCdr1p suggests that there are either common (to R6G) or independent binding sites of FAR on CaCdr1p. However, elaborate binding studies will be required to resolve this issue.

When a nontoxic concentration of FAR was used in combination, it displayed synergy with the drugs in Candida cells. Notably, FAR could also synergize with the drugs in azole-resistant (with high MIC₈₀ values) Candida isolates. It should be pointed out that the modulator effect of FAR over the efflux of drugs mediated by ABC multidrug transporter proteins was independent of its ability to show synergism with the drugs. For example, the synergistic action of nontoxic FAR (100 μM/22 μg ml⁻¹) in combination with the nonlethal concentration of drugs was associated with the substantial accumulation of ROS levels (Fig. 7A), which was not the case when FAR alone at its modulator concentration was used. Our observation that FAR acts synergistically with the tested drugs suggests a possibility wherein the combination could be used to inhibit biofilm development in C. albicans. However, this warrants further studies on biofilm development at the synergistic concentrations of FAR and drugs. We had earlier shown that CUR acts as an antifungal agent against C. albicans and is synergistic to azoles (FLC) or polyenes (AMB) via generation of ROS and induction of PCD of C. albicans cells (28). Thus, various effects of CUR seem to mimic the action of FAR. However, we observed that the CUR effect was independent of FAR levels. In a mutant strain of C. albicans which was a knockout of dpp3 (Δdpp3 mutant), which encodes a phosphatase for converting farnesyl pyrophosphate to FAR, polyphenol CUR continued to inhibit cell growth, which could be reversed by the addition of an antioxidant (28). Collectively, we demonstrate that FAR is a substrate-specific modulator of efflux of drugs mediated by ABC transporter proteins and, if given in combination, is synergistic to drugs and accumulates ROS, resulting in early PCD.