In the present study, we have investigated the antifungal effects of a natural polyphenol, CUR (curcumin), against albicans and non-albicans species of Candida and have shown its ability to inhibit the growth of all the tested strains. The inhibitory effects of CUR were independent of the status of the multidrug efflux pump proteins belonging to either ABC transporter (ATP-binding cassette transporter) or MFS (major facilitator) superfamilies of transporters. By using a systemic murine model of infection, we established that CUR and piperine, when administered together, caused a significant fungal load reduction (1.4log10) in kidneys of Swiss mice. Additionally, CUR raised the levels of ROS (reactive oxygen species), which, as revealed by annexin V–FITC labelling, triggered early apoptosis in Candida cells. Coincident with the raised ROS levels, mRNAs of tested oxidative stress-related genes [CAP1 (Candida albicans AP-1), CaIPF7817 (putative NADH-dependent flavin oxidoreductase), SOD2 (superoxide dismutase 2), GRP2 (NADPH-dependent methyl glyoxal reductase) and CAT1 (catalase 1)] were also elevated. The growth inhibitory effects of CUR could be reversed by the addition of natural and synthetic antioxidants. Notably, independent of ROS status, polyphenol CUR prevented hyphae development in both liquid and solid hypha-inducing media by targeting the global suppressor TUP1 (thymidine uptake 1). Taken together, our results provide the first evidence that CUR acts as an antifungal agent, via generation of oxidative stress, and inhibits hyphae development by targeting TUP1.

INTRODUCTION

The dimorphic opportunistic pathogen Candida albicans is normally a commensal organism in humans, but when the host is unable to mount an adequate immune response, such as in AIDS, organ transplantation, diabetes, burn or cancer patients, it results in mucosal, cutaneous or invasive mycosis [1]. Infections caused by C. albicans are commonly treated either by azoles or by non-azole antifungal agents. Widespread and prolonged usage of antifungals, in recent years, has led to the emergence of azole-resistant strains of Candida, which display MDR (multidrug resistance) [2]. Various mechanisms that contribute to the development of azole resistance have been reported, which include overexpression of or point mutations in ERG11 and the target enzyme of azoles, i.e. lanosterol 14α-demethylase [2], and an overexpression of the drug efflux pump encoding genes, namely CaCDR1 (C. albicans Candida drug resistance 1), CaCDR2 and CaMDR1 (C. albicans MDR1) belonging to the ABC transporter (ATP-binding cassette transporter) and MFS (major facilitator) superfamilies of transporters respectively [2].

Although mechanisms of antifungal resistance and major factors that contribute to it are fairly established, there is evidence suggesting that MDR is a multifactorial phenomenon, which originates from as yet unknown mechanisms. For example, morphological regulators such as Δefg1 (Prasad, T., Hameed, S., Manoharlal, R., Biswas, S., Mukhopadhyay, C. K., Goswami, S. K. and Prasad, R., unpublished work), a homologue of the bacterial two-component response regulator Δssk1, and iron deprivation show enhanced sensitivity to drugs in C. albicans [4,5]. There are azole-resistant clinical isolates of C. albicans where mechanisms of resistance appear to be different from the commonly known strategies adopted by Candida [6].

The present study deals with the evaluation of antifungal activity of a natural plant polyphenol, CUR (curcumin), produced by the rhizome of Curcuma longa. CUR, which is an important spice in the Asian diet, has several important pharmacological properties, notably, antioxidant, antimutagenetic and antitumour activities [7]. It could block HIV-1 replication by inhibiting the activity of its LTR (long terminal repeat) and synergistically works with dideoxyinosine, a reverse transcriptase inhibitor in HIV-1 cells [8]. CUR alters cellular redox homoeostasis, and disrupts mitochondrial function in cultured, transformed cells [9,10].

In the present study, we show that CUR can be lethal to C. albicans as well as to non-albicans species, increases ROS (reactive oxygen species) levels and brings about early apoptosis, which could be reversed by the addition of antioxidants in C. albicans cells.

MATERIALS AND METHODS

Materials

Commercial-grade mixture of curcuminoids, commonly known as CUR, DCFH-DA (2′,7′-dichlorofluorescein diacetate), PDTC (pyrrolidinedithiocarbamate), AA (ascorbic acid), PEG [poly(ethylene glycol)], PIP (piperine) and other molecular grade chemicals were obtained from Sigma Chemicals (St. Louis, MO, U.S.A.). An annexin V–FITC apoptosis detection kit was obtained from BD Biosciences. Zymolyase 100T was purchased from Seikagaku Corporation. The oligonucleotides used in the present study were commercially synthesized by Sigma–Aldrich.

Yeast strains and growth media

Strains used in the present study are listed in Supplementary Table S1 (http://www.bioscirep.org/bsr/030/bsr0300391add.htm). The yeast strains were cultured in YEPD (yeast extract/peptone/dextrose) broth (BIO101; Vista). For agar plates, 2.5% (w/v) bacto agar (Difco, BD Biosciences) was added to the medium. All strains were stored as frozen stock with 15% (v/v) glycerol at –80°C. Before each experiment, cells were freshly revived on YEPD plates from the stock.

In vivo antifungal susceptibility testing

A C. albicans strain (ATCC 36082) was grown overnight on YEPD at 30°C and suspended in sterile normal saline to adjust the D600 (attenuance at 600 nm) to 1.0. The final inoculum was prepared by 1:20 dilution of the original suspension. The cfu (colony-forming units) per ml of inoculum was 5.5×107. CUR was dissolved in 20% (v/v) PEG, whereas FLC (fluconazole) and PIP were dissolved in sterile water. Swiss albino mice (n=6) of either sex weighing 20±2 g were procured from an in-house facility. Animals were taken 2–3 days before the start of experiments to acclimatize to the experimental environment. Feed and water were provided ad libitum during the entire study. We have complied with the ethical standards that were approved by the institutional ethics committee. All mice were infected with 200 μl of the cell suspension (1×107 cfu) by the intravenous route. Treatment started 1 h post-infection: in one group, CUR alone (100 mg/kg of body weight) was intraperitoneally administered; a second group was dosed with CUR plus PIP (100 and 20 mg/kg of body weight respectively) administered orally; whereas in the third group, reference standard FLC was given (50 mg/kg of body weight, orally). The fourth group was kept as untreated control and was administered vehicle (20% PEG, orally) only. In all the groups, a second dose was administered 6 h after the first dose and animals were treated for 2 days. The data were analysed by using GraphPad Prism, version 5.1. The limit of detection for live C. albicans was 1.7log10.

Time kill assays

C. albicans cells at a concentration of 103 cfu/ml were inoculated in YEPD medium containing either CUR (185 mg/l) or antioxidants PDTC (10 μM)/AA (25 mM) alone or a combination of both CUR and antioxidants. At predetermined time points (0, 4, 8, 12, 16, 20 and 24 h, at 30°C incubation; agitation 200 rev./min), a 100 μl aliquot was removed, serially diluted (10-fold) in 0.9% saline and plated on to YEPD agar plates. Colony counts were determined after incubation at 30°C for 48 h [11].

Measurement of ROS production

Endogenous amounts of ROS were measured by a fluorimetric assay with DCFH-DA [12]. Briefly, the cells were adjusted to a D660 of 1 in 10 ml of PBS and centrifuged at 2500 g for 15 min. The cell pellet was then resuspended in PBS and treated with appropriately diluted PDTC or AA for 1 h or was left untreated at room temperature (25°C). After incubation with CUR at 37°C for different time intervals as indicated, 10 μM DCFH-DA in PBS was added. The FIs (fluorescence intensities) (λex=485 nm and λem=540 nm) of the resuspended cells were measured with a spectrofluorimeter (Varian, Cary Eclipse) and the images of DCF fluorescence were taken by using a fluorescence microscope (Carl Zeiss).

Analysis of apoptotic markers

Protoplasts of C. albicans were stained with propidium iodide and FITC-labelled annexin V by using the annexin V–FITC apoptosis detection kit (BD Biosciences) to assess the cellular integrity and the externalization of PS (phosphatidylserine) as described earlier [13]. The cells were analysed by using an FACS® Caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, U.S.A.) using λex=488 nm excitation and a 515 nm band pass filter for FITC detection and a filter >560 nm for propidium iodide detection. A total of 10000 events were counted at the flow rate. Data analysis was performed using Cell Quest software (Becton Dickinson Immunocytometry Systems) [13,14].

Morphological studies

To check the hypha status, the cells were grown in the presence/absence of CUR alone or CUR+PDTC in liquid or solid (2.5% agar) YEPD with 10% (v/v) FBS (fetal bovine serum) or in liquid spider medium and incubated for 6 h (in liquid medium) or for 3 days (in the case of solid medium) at 37°C. Colony morphologies on solid plates and filamentation in liquid medium were analysed microscopically (Carl Zeiss) [15].

RNA isolation and hybridization

Total RNA from the mid-exponentially grown C. albicans cells was prepared in the presence/absence of CUR [5]. Approx. 25 μg of total RNA from the above samples was hybridized with probes derived from genespecific sequences as mentioned in Supplementary Table S2 (http://www.bioscirep.org/bsr/030/bsr0300391add.htm). Hybridization signal intensity was quantified with a phosphoimager scanner (FLA-5000; Fuji phosphoimager).

RESULTS

CUR inhibits the growth of Candida cells

To investigate the antifungal effect of CUR on C. albicans cells, we used a commercial preparation of CUR, which is a mixture of three major curcuminoids, namely diferuloylmethane (CUR I), demethoxycurcumin (CUR II) and bisdemethoxycurcumin (CUR III), and contains predominantly CUR I (~77%) followed by CUR II (17%) and CUR III (3%) [7]. For this, we employed spot and broth microdilution drug susceptibility assays. CUR over a range of concentrations was able to inhibit the growth of cells after concentrations ≥185 mg/l in a broth microdilution assay, whereas higher concentrations (296–370 mg/l) were needed to inhibit the growth in solid medium (Figures 1A and 1B). The growth inhibitory effect of CUR was also evident with non-albicans species. For example, on solid medium, CUR at ≥46.25 mg/l was able to inhibit the growth of Candida tropicalis Candida dubliniensis and Candida utilis whereas ≥92.5 and ≥185 mg/l of it was needed to inhibit the growth of Candida kefyr and Candida krusei respectively. Both Candida parapsilosis and Candida glabrata required 370 mg/l CUR to show growth inhibition (Figures 1A and 1B). All the three purified curcuminoids from CUR did inhibit the growth of Candida cells, which was comparable with a commercial preparation of CUR (results not shown). For all the subsequent experiments, commercial CUR was used.

Effect of CUR on the growth of Candida cells

Figure 1
Effect of CUR on the growth of Candida cells

(A) The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) Determination of growth inhibition of the Candida cells by the broth microdilution assay in the presence of CUR at concentrations ranging from 1.44 to 740 mg/l. Growth of the cells was evaluated both visually and by reading the attenuance (D600) in a microtitre reader as described earlier [44]. A stock solution of 11 mg/ml was used (DMSO). Growth was not affected by the presence of the solvent (results not shown). Filled/empty triangle, filled/empty square, filled/empty diamond and filled/empty circle represent C. krusei, C. albicans, C. tropicalis, C. dubliniensis, C. kefyr, C. utilis, C. parapsilosis and C. glabrata respectively.

Figure 1
Effect of CUR on the growth of Candida cells

(A) The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) Determination of growth inhibition of the Candida cells by the broth microdilution assay in the presence of CUR at concentrations ranging from 1.44 to 740 mg/l. Growth of the cells was evaluated both visually and by reading the attenuance (D600) in a microtitre reader as described earlier [44]. A stock solution of 11 mg/ml was used (DMSO). Growth was not affected by the presence of the solvent (results not shown). Filled/empty triangle, filled/empty square, filled/empty diamond and filled/empty circle represent C. krusei, C. albicans, C. tropicalis, C. dubliniensis, C. kefyr, C. utilis, C. parapsilosis and C. glabrata respectively.

Co-administration of CUR and PIP reduces the Candida load in vivo

CUR has poor bioavailability owing to its rapid metabolism in liver and intestinal walls. Co-administration of PIP, an inhibitor of hepatic and intestinal glucuronidation, is known to enhance the bioavailability of CUR [16]. To determine the in vivo antifungal activity of CUR against C. albicans (ATCC 36082), a systemic murine model of infection was employed [17]. As depicted in Figure 2, the effect of CUR was evaluated by comparing the live Candida load reduction in the kidneys of treated and untreated mice after administration of CUR or CUR+PIP. It was observed that administration of CUR (intraperitoneally) alone led to an insignificant fungal load decrease of 1.0log10 with a P value of 0.0887. However, the administration of CUR along with PIP (orally) causes a significant fungal load reduction of 1.4log10 with a P value of 0.0199. In the case of positive control FLC, there was a fungal load reduction of 1.9log10, with a P value of 0.0029. Notably, the MIC80 (lowest drug concentration that causes 80% inhibition of fungal growth) value of C. albicans (ATCC 36082) was similar to that of SC5314 (ATCC MYA2876) used in the present study for the subsequent experiments (results not shown).

Kidney cfu assay of mice with systemic candidiasis

Figure 2
Kidney cfu assay of mice with systemic candidiasis

(A) In vivo efficacy of CUR against C. albicans (strain 36082) in the murine systemic infection model. Swiss albino mice (n=6) of either sex weighing 20±2 g were used in four experimental groups. In one group, CUR was administered 100 mg/kg of body weight (intraperitonealy, IP), the second group was dosed with CUR (100 mg/kg) in combination with PIP (20 mg/kg) administered concomitantly orally (oral administration, PO), and the reference standard FLC (50 mg/kg, orally) was given to the third group. The fourth group was kept as untreated control and was administered vehicle (20% PEG and water) only. ‘Limit’ indicates the minimum level of detection for live C. albicans. (B) The Table presents the mean log10 cfu of C. albicans in the kidney of control (untreated) and treated groups. Statistical analysis was performed using a t test and the results were considered significant when P values were less than 0.05.

Figure 2
Kidney cfu assay of mice with systemic candidiasis

(A) In vivo efficacy of CUR against C. albicans (strain 36082) in the murine systemic infection model. Swiss albino mice (n=6) of either sex weighing 20±2 g were used in four experimental groups. In one group, CUR was administered 100 mg/kg of body weight (intraperitonealy, IP), the second group was dosed with CUR (100 mg/kg) in combination with PIP (20 mg/kg) administered concomitantly orally (oral administration, PO), and the reference standard FLC (50 mg/kg, orally) was given to the third group. The fourth group was kept as untreated control and was administered vehicle (20% PEG and water) only. ‘Limit’ indicates the minimum level of detection for live C. albicans. (B) The Table presents the mean log10 cfu of C. albicans in the kidney of control (untreated) and treated groups. Statistical analysis was performed using a t test and the results were considered significant when P values were less than 0.05.

Antifungal effect of CUR is independent of the drug efflux pump

When the drug efflux pump (CaCDR1/CaCDR2/CaMDR1) [1820] null mutants were grown in the presence of various concentrations of CUR, the drug susceptibility pattern remained similar to the wild-type strain (Figure 3A). The clinical matched pair of AS (azole-susceptible) and AR (azole-resistant) isolates, which show increased resistance to azoles owing to an overexpression of either CaCDR1(Gu4, Gu5) [21] or CaMDR1 (F2, F5) [22] genes, remained sensitive to CUR (Figure 3B). CUR did not affect the expression of genes encoding MDR pump proteins (Figure 3C).

Antifungal effects of CUR against C. albicans, in cells either lacking or overexpressing the drug efflux pumps

Figure 3
Antifungal effects of CUR against C. albicans, in cells either lacking or overexpressing the drug efflux pumps

The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (A) Null mutants of C. albicansCaCdr1, ΔCaCdr2, ΔCaMdr1) lacking functional drug transporters. (B) Matched pair isolates of C. albicans (Gu4 and Gu5) overexpressing the transporter CaCDR1 or CaMDR1 (F2 and F5). Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (C) Transcript levels of CDR1/CDR2/CaMDR1 in the wild-type strain SC5314 in the absence and presence of CUR (185 mg/l, 16 h). ACT1 mRNA levels were used as a loading control.

Figure 3
Antifungal effects of CUR against C. albicans, in cells either lacking or overexpressing the drug efflux pumps

The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (A) Null mutants of C. albicansCaCdr1, ΔCaCdr2, ΔCaMdr1) lacking functional drug transporters. (B) Matched pair isolates of C. albicans (Gu4 and Gu5) overexpressing the transporter CaCDR1 or CaMDR1 (F2 and F5). Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (C) Transcript levels of CDR1/CDR2/CaMDR1 in the wild-type strain SC5314 in the absence and presence of CUR (185 mg/l, 16 h). ACT1 mRNA levels were used as a loading control.

Oxidative stress null mutants are susceptible to CUR

To understand the molecular basis of antifungal activity of CUR, we evaluated its effect on various categories of mutants of C. albicans. These included morphological nulls Δtup1 (Tup1p, which is a transcriptional co-repressor and represses filamentous growth [23]), Δefg1 (Efg1p, a transcriptional repressor required for hyphal growth [24]), Δcph1 (Cph1p, a transcription factor required for mating and hyphal growth on solid medium [24]), Δnrg1 (Nrg1p, a transcription repressor, regulates hyphal genes and virulence genes [23]), Δras1 {Ras1p, which is a RAS signal transduction GTPase and regulates cAMP and MAPK (mitogen-activated protein kinase) pathways [25]}, Δssk1 (Ssk1p, which is a response regulator of the two-component system and plays a role in cell wall biosynthesis and virulence [26]), oxidative stress nulls ΔCaIpf7817 (CaIpf7817p, which is involved in the regulation of redox homoeostasis [27]), ΔCap1 (Cap1p, which is a transcription factor and regulates the oxidative stress response [28,29]), Δtac1 (Tac1p, a transcription factor involved in the up-regulation of CDR1 and CDR2 [30]), Δftr1 (Ftr1p, a high-affinity iron permease [31]) and Δccc2 (Ccc2p, a copper-transporting ATPase) [32]. Notably, only the oxidative stress nulls ΔCaIpf7817 and ΔCap1 appeared to be highly susceptible to CUR in comparison with the wild-type strain (Figure 4A). As depicted in Figure 4(B), there was a significant reduction in the MIC80 values of the oxidative stress mutants when grown in the presence of CUR as compared with the wild-type strain. None of the other tested mutants showed any difference in the MIC80 values when grown on CUR (see Supplementary Table S3 at http://www.bioscirep.org/bsr/030/bsr0300391add.htm).

Susceptibility of oxidative stress mutants of C. albicans to CUR

Figure 4
Susceptibility of oxidative stress mutants of C. albicans to CUR

(A) The cells (SC5314/ΔCap1CaIpf7817) were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) Determination of growth inhibition of the Candida cells by the broth microdilution assay in the presence of CUR at concentrations ranging from 1.44 to 740 mg/l. Growth of the cells was evaluated both visually and by reading the D600 in a microtitre reader as described earlier [44]. Filled/empty squares and filled triangles represent SC5314/ΔCap1 and ΔCaIpf7817 respectively. The inset shows the MIC80 values. A stock solution of 11 mg/ml was used (DMSO). Growth was not affected by the presence of the solvent (results not shown).

Figure 4
Susceptibility of oxidative stress mutants of C. albicans to CUR

(A) The cells (SC5314/ΔCap1CaIpf7817) were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) Determination of growth inhibition of the Candida cells by the broth microdilution assay in the presence of CUR at concentrations ranging from 1.44 to 740 mg/l. Growth of the cells was evaluated both visually and by reading the D600 in a microtitre reader as described earlier [44]. Filled/empty squares and filled triangles represent SC5314/ΔCap1 and ΔCaIpf7817 respectively. The inset shows the MIC80 values. A stock solution of 11 mg/ml was used (DMSO). Growth was not affected by the presence of the solvent (results not shown).

Antifungal effects of CUR could be reversed by antioxidants

We further confirmed the role of oxidative stress by performing spot assays in the presence of CUR and antioxidants such as PDTC or AA. For this, we used a range of antioxidant concentrations (5–100 μM for PDTC and 25–100 mM for AA) and observed that 10 μM of PDTC or 25 mM of AA could only partially restore the CUR effect. A higher concentration of each antioxidant was toxic to cells (results not shown). As depicted in Figure 5(A), addition of PDTC (10 μM) or AA (25 mM) alone had no effect on the growth of cells but, when added along with CUR (296 mg/l), the growth inhibition was reversed. This reversal of growth inhibition due to antioxidants was further confirmed by the colony formation assay. As compared with CUR alone, which killed ~96% of the cells, the percentage of viable cells in the presence of CUR and antioxidants was considerably increased (Figure 5B).

Spot assays in the presence of antioxidants/CUR and the time kill assays

Figure 5
Spot assays in the presence of antioxidants/CUR and the time kill assays

(A) The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of CUR (296 mg/l) alone or CUR and antioxidants (10 μM PDTC or 25 mM AA). The antioxidants have no effect on the growth of cells. Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (B) Time kill curves of the wild-type strain of C. albicans in the presence of CUR/PDTC/AA alone or in combination were obtained by using initial inoculums of 103 cfu/ml. The filled/empty diamonds, squares and circles represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values shown are the means and S.D. (indicated by error bars) for three independent experiments.

Figure 5
Spot assays in the presence of antioxidants/CUR and the time kill assays

(A) The cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of CUR (296 mg/l) alone or CUR and antioxidants (10 μM PDTC or 25 mM AA). The antioxidants have no effect on the growth of cells. Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (B) Time kill curves of the wild-type strain of C. albicans in the presence of CUR/PDTC/AA alone or in combination were obtained by using initial inoculums of 103 cfu/ml. The filled/empty diamonds, squares and circles represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values shown are the means and S.D. (indicated by error bars) for three independent experiments.

CUR generates ROS

In the following experiments, we analysed whether ROS formation could be a key event in the CUR-induced killing of C. albicans. Using a fluorimetric assay, we could demonstrate that in the presence of CUR, there was an increase in FI, which coincided with induced endogenous ROS in Candida (Figures 6A and 6B). The net ROS production in cells raised by CUR could be reversed by the addition of antioxidant PDTC or AA. The ROS levels in non-albicans species were also raised to different levels when CUR was present in growth medium, which could be reversed by the addition of antioxidants (Figure 6C). It should be pointed out that PDTC and AA alone in the growth medium inhibited the basal level of ROS production between 10 and 20% (Figure 6B). Expectedly, the highly CUR-susceptible oxidative stress mutants ΔCaIpf7817 and ΔCap1 displayed 3–5 times higher basal ROS levels, which was further increased by CUR in a reversible manner (Figure 7A). As depicted in Figure 7(B), the presence of CUR increased the transcript levels of the genes [CAP1 (C. albicans AP-1), CaIPF7817 (putative NADH-dependent flavin oxidoreductase), GRP2 (NADPH-dependent methyl glyoxal reductase), CAT1 (catalase 1) and SOD2 (superoxide dismutase 2)] associated with maintenance of oxidative stress.

ROS levels in the presence of CUR in C. albicans cells

Figure 6
ROS levels in the presence of CUR in C. albicans cells

(A) Images of DCF fluorescence due to CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA) treatment were taken with a fluorescence microscope (Carl Zeiss). (B) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells. The fluorescence emitted by the cells was measured by using a spectrofluorimeter (Varian, Cary Eclipse; λex=485 and λem=540 nm). The filled/empty diamond, filled/empty square and filled/empty circle represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The bar graph shows the level of ROS produced at 24 h of administration of CUR. (C) ROS produced in non-albicans species of Candida on CUR (185 mg/l) or CUR+PDTC or CUR+AA (10 μM PDTC and 25 mM AA) treatment (16 h). The values are the means and S.D. (indicated by error bars) for three independent experiments.

Figure 6
ROS levels in the presence of CUR in C. albicans cells

(A) Images of DCF fluorescence due to CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA) treatment were taken with a fluorescence microscope (Carl Zeiss). (B) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells. The fluorescence emitted by the cells was measured by using a spectrofluorimeter (Varian, Cary Eclipse; λex=485 and λem=540 nm). The filled/empty diamond, filled/empty square and filled/empty circle represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The bar graph shows the level of ROS produced at 24 h of administration of CUR. (C) ROS produced in non-albicans species of Candida on CUR (185 mg/l) or CUR+PDTC or CUR+AA (10 μM PDTC and 25 mM AA) treatment (16 h). The values are the means and S.D. (indicated by error bars) for three independent experiments.

ROS levels in the oxidative stress mutants and mRNA transcript levels in C. albicans cells

Figure 7
ROS levels in the oxidative stress mutants and mRNA transcript levels in C. albicans cells

(A) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells, namely wild-type (SC5314), ΔCap1 and ΔCaIpf7817. The fluorescence emitted by the cells was measured by using a fluorescence spectrometer (λex=485 nm and λex=540 nm). Bars with dots, diagonal lines, weave, horizontal lines, bricks and checker board represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values are the means and S.D. (indicated by error bars) for three independent experiments. (B) Transcript levels of CAP1, CaIPF7817, SOD2, GRP2 and CAT1 in the wild-type strain SC5314 in the presence and absence of CUR (185 mg/l, 4 h). ACT1 mRNA levels were used as a loading control.

Figure 7
ROS levels in the oxidative stress mutants and mRNA transcript levels in C. albicans cells

(A) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells, namely wild-type (SC5314), ΔCap1 and ΔCaIpf7817. The fluorescence emitted by the cells was measured by using a fluorescence spectrometer (λex=485 nm and λex=540 nm). Bars with dots, diagonal lines, weave, horizontal lines, bricks and checker board represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values are the means and S.D. (indicated by error bars) for three independent experiments. (B) Transcript levels of CAP1, CaIPF7817, SOD2, GRP2 and CAT1 in the wild-type strain SC5314 in the presence and absence of CUR (185 mg/l, 4 h). ACT1 mRNA levels were used as a loading control.

CUR induces apoptosis in Candida cells

Translocation of PS to the outer monolayer of the lipid bilayer of the PM (plasma membrane) is an early marker of apoptosis [13,14]. We explored whether CUR induces apoptosis in C. albicans by measuring PS externalization using the annexin V–FITC assay. As depicted in Figure 8(A), after 4 h of incubation of cells with CUR, the population of cells had 22.88% exposed PS as compared with 3.17% in untreated cells (Figure 8A, panels II and I respectively). Notably the CUR-induced externalization of PS could be arrested to 2.28 or 3.77% if cells were pretreated with antioxidant PDTC or AA before the incubation with CUR (Figure 8A, panels III and IV respectively).

Externalization of PS in CUR-treated cells

Figure 8
Externalization of PS in CUR-treated cells

Panel I represents untreated, stained C. albicans (SC5314) cells, panel II shows CUR-treated cells, panels III and IV are the PDTC/AA pretreated cells in the presence of CUR and stained with (A) annexin V–FITC alone or (B) co-stained with annexin V–FITC and propidium iodide. For treatment, the cells were incubated with CUR (185 mg/l, 4 h) and analysed by flow cytometry as described in the Materials and methods section. The lower right quadrants of the various panels represent early apoptotic cells, and the upper right quadrants represent late apoptotic or necrotic cells. (C) Transcript level of CaMCA1 in the wild-type strain SC5314 in the absence and presence of CUR (185 mg/l, 4 h). ACT1 mRNA levels were used as a loading control.

Figure 8
Externalization of PS in CUR-treated cells

Panel I represents untreated, stained C. albicans (SC5314) cells, panel II shows CUR-treated cells, panels III and IV are the PDTC/AA pretreated cells in the presence of CUR and stained with (A) annexin V–FITC alone or (B) co-stained with annexin V–FITC and propidium iodide. For treatment, the cells were incubated with CUR (185 mg/l, 4 h) and analysed by flow cytometry as described in the Materials and methods section. The lower right quadrants of the various panels represent early apoptotic cells, and the upper right quadrants represent late apoptotic or necrotic cells. (C) Transcript level of CaMCA1 in the wild-type strain SC5314 in the absence and presence of CUR (185 mg/l, 4 h). ACT1 mRNA levels were used as a loading control.

Since after the loss of membrane integrity, annexin V–FITC also labels necrotic cells, simultaneous addition of propidium iodide, which does not permeate cells with an intact PM, allows discrimination between apoptotic (annexin V positive, propidium iodide negative), necrotic (both annexin V and propidium iodide positive) and live (both annexin V and propidium iodide negative) cells [13,14]. Accordingly, we examined the effect of CUR on the overall population distribution between apoptotic and necrotic cells. For this, CUR (185 mg/l)-treated cells were double stained with annexin V–FITC and propidium iodide. As shown in Figure 8(B) (panel II), a significant percentage (28.95%, lower right quadrants) of cells stained positive for annexin V–FITC as compared with none (Figure 8, panel I) in untreated cells. It was confirmed from the double staining (annexin V–FITC and propidium iodide, Figure 8B, panel II, lower right quadrants), that the single stained (annexin V–FITC) cells represents only apoptotic cells (Figure 8A, panel II, lower right quadrants). The late apoptotic/necrotic cells increased to 11.95% in 4 h (Figure 8B, panel II, upper right quadrant) when treated with CUR, which could be reversed by the antioxidants such as PDTC or AA (Figure 8B, panels III and IV, upper right quadrants).

MCA1 (metacaspase 1) encodes a homologue of a mammalian caspase in C. albicans [33]. To determine the involvement of caspases in CUR-induced cytotoxicity in C. albicans, the expression of CaMCA1 was determined in exponential phase cells by Northern-blot analysis. It was observed that after a 4 h exposure to CUR, the CaMCA1 transcript level was also increased when compared with untreated cells (Figure 8C).

CUR inhibits hyphae development by Candida

Mycelial development of C. albicans is influenced by many factors and is controlled by well-known morphological regulators. In the present study, we observed that CUR (37 mg/l) when added to hypha-inducing solid or liquid medium prevented the development of hyphae of C. albicans cells. In contrast with the other effects of CUR, the inhibition of hyphae development could not be reversed by the addition of an antioxidant such as PDTC (10 μM) or AA (25 mM) (Figure 9).

Hyphae development in the presence and absence of CUR/CUR+ PDTC in solid and liquid hypha-inducing media

Figure 9
Hyphae development in the presence and absence of CUR/CUR+ PDTC in solid and liquid hypha-inducing media

Response to CUR (37 mg/l) and CUR (37 mg/l)+PDTC (10 μM) by C. albicans (SC5314) cells under conditions that promote hyphal growth. The cells were grown in the absence or presence of CUR alone or CUR+PDTC in (A) solid YEPD (2.5% agar) with 10% FBS or in (B) liquid spider medium. Cells were incubated for 6 h in liquid medium and for 3 days in solid medium at 37°C. Colony morphologies on solid plates and filamentation in liquid medium were examined microscopically (Carl Zeiss) as mentioned in the Materials and methods section.

Figure 9
Hyphae development in the presence and absence of CUR/CUR+ PDTC in solid and liquid hypha-inducing media

Response to CUR (37 mg/l) and CUR (37 mg/l)+PDTC (10 μM) by C. albicans (SC5314) cells under conditions that promote hyphal growth. The cells were grown in the absence or presence of CUR alone or CUR+PDTC in (A) solid YEPD (2.5% agar) with 10% FBS or in (B) liquid spider medium. Cells were incubated for 6 h in liquid medium and for 3 days in solid medium at 37°C. Colony morphologies on solid plates and filamentation in liquid medium were examined microscopically (Carl Zeiss) as mentioned in the Materials and methods section.

Hyphae development by tup1 nulls could not be inhibited by CUR

We examined the effect of CUR (37 mg/l) in several null mutants lacking morphological transcription regulators and found that CUR could not prevent hyphae development in Δtup1 cells [34] (Figure 10A). This coincided with a raised TUP1 (thymidine uptake 1) transcript level due to CUR in a wild-type strain of C. albicans (Figure 10B).

Effect of CUR on the hyphae development of Δtup1 cells

Figure 10
Effect of CUR on the hyphae development of Δtup1 cells

(A) Response to CUR and CUR+PDTC by C. albicans under conditions that promote hyphal growth. The cells were grown in the presence or absence of CUR alone (37 mg/l) or CUR (37 mg/l)+PDTC (10 μM) in YEPD with 10% FBS and incubated at 37°C for 6 h. The filamentation in liquid medium was examined microscopically (Carl Zeiss). (B) Transcript levels of Tup1 in the wild-type strain SC5314 in the presence and absence of CUR (37 mg/l) at the indicated time points. Constitutively expressing ACT1 transcript was used as a loading control. (C) Quantification of Northern-blot hybridization.

Figure 10
Effect of CUR on the hyphae development of Δtup1 cells

(A) Response to CUR and CUR+PDTC by C. albicans under conditions that promote hyphal growth. The cells were grown in the presence or absence of CUR alone (37 mg/l) or CUR (37 mg/l)+PDTC (10 μM) in YEPD with 10% FBS and incubated at 37°C for 6 h. The filamentation in liquid medium was examined microscopically (Carl Zeiss). (B) Transcript levels of Tup1 in the wild-type strain SC5314 in the presence and absence of CUR (37 mg/l) at the indicated time points. Constitutively expressing ACT1 transcript was used as a loading control. (C) Quantification of Northern-blot hybridization.

CUR works independently of the quorum sensing molecule farnesol

It is known that the quorum sensing molecule farnesol raises ROS levels and inhibits hyphae development by targeting the repressor TUP1 [34]. We observed that polyphenol CUR apparently mimics the farnesol affect. To establish the link between CUR and farnseol pathways, we examined the various effects of CUR, namely antifungal, ROS generation and the inhibitory effect on mycelial development in a C. albicans strain defective in farnesol production. DPP3 (diacylglycerol pyrophosphate phosphatase) encodes a phosphatase that converts farnesyl pyrophosphate into farnesol. It is reported that DPP3 knockout (KWN2) produces six times less farnesol in comparison with the parent strain (SN152) [35]. Figure 11(A) shows that CUR could inhibit the growth of the DPP3 null strain similarly to the wild-type strain. Additionally, the growth inhibition of Δdpp3 cells by CUR could be reversed by antioxidants (Figure 11B). Similarly, the CUR effect to raise ROS levels and to inhibit hyphae formation remained unaffected in Δdpp3 cells (Figures 11C and 11D).

Effects of CUR on Δdpp3 cells

Figure 11
Effects of CUR on Δdpp3 cells

(A) The wild-type (SN152) and Δdpp3 (KWN2) cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of CUR (296 mg/l) alone or CUR and antioxidants (10 μM PDTC or 25 mM AA). The antioxidants (10 μM PDTC or 25 mM AA) have no effect on the growth of cells. Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (C) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells, namely wild-type (SN152) and Δdpp3 (KWN2) cells. The fluorescence emitted by the cells was measured by using a fluorescence spectrometer (λex=485 and λem=540 nm). Bars with dots, diagonal lines, weave, horizontal lines, bricks and checker board represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values are the means and S.D. (indicated by error bars) for three independent experiments. (D) Response to CUR and CUR+PDTC by C. albicans under conditions that promote hyphal growth. The cells were grown in the presence or absence of CUR alone (37 mg/l) or CUR (37 mg/l)+PDTC (10 μM) in YEPD with 10% FBS and incubated at 37°C for 6 h. The filamentation in liquid medium was examined microscopically (Carl Zeiss).

Figure 11
Effects of CUR on Δdpp3 cells

(A) The wild-type (SN152) and Δdpp3 (KWN2) cells were grown overnight on YEPD plates and then resuspended in normal saline to a D600 of 0.1. A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of various concentrations of CUR (37–370 mg/l). (B) A 5 μl portion of 5-fold serial dilution of each strain was spotted on to YEPD plates as described earlier [44], either in the absence or presence of CUR (296 mg/l) alone or CUR and antioxidants (10 μM PDTC or 25 mM AA). The antioxidants (10 μM PDTC or 25 mM AA) have no effect on the growth of cells. Growth differences were evaluated after 48 h of incubation as mentioned earlier [44]. (C) Amounts of ROS produced in CUR (185 mg/l) or CUR+antioxidants (10 μM PDTC or 25 mM AA)-treated cells, namely wild-type (SN152) and Δdpp3 (KWN2) cells. The fluorescence emitted by the cells was measured by using a fluorescence spectrometer (λex=485 and λem=540 nm). Bars with dots, diagonal lines, weave, horizontal lines, bricks and checker board represent cells alone, cells+PDTC, cells+CUR, cells+CUR+PDTC, cells+AA and cells+CUR+AA respectively. The values are the means and S.D. (indicated by error bars) for three independent experiments. (D) Response to CUR and CUR+PDTC by C. albicans under conditions that promote hyphal growth. The cells were grown in the presence or absence of CUR alone (37 mg/l) or CUR (37 mg/l)+PDTC (10 μM) in YEPD with 10% FBS and incubated at 37°C for 6 h. The filamentation in liquid medium was examined microscopically (Carl Zeiss).

DISCUSSION

In the present study, we have investigated the antifungal effects of a natural polyphenol CUR against albicans and non-albicans species of Candida and have shown that the growth of all the tested strains of Candida could be inhibited by CUR (Figure 1). While demonstrating the anticancer effects of CUR, it has been observed that the systemic exposure to CUR remains too low to exhibit sufficient pharmacological activity. However, concomitant administration of PIP increases bioavailability of CUR both in humans and in rats [7,16]. On the basis of these findings, PIP, a known inhibitor of hepatic and intestinal glucuronidation, was combined with CUR and administered to check its in vivo efficacy. Indeed, when a combination of CUR and PIP was administered, there was a significant and higher reduction in fungal load (1.4log10) in kidneys of Swiss mice as compared with the case when CUR was administered alone (1log10) (Figure 2). Pharmacokinetic studies have indicated that after oral administration in rats and humans, CUR is transformed into metabolites like CUR glucuronides [7]. PIP remarkably enhances the bioavailability of CUR in mice, resulting in significant reduction in fungal load; this suggests that these metabolites may also contribute to the antifungal effect of CUR. Elaborate in vivo studies are required to find out the efficacy across the fungal species. Also, research efforts are necessary to increase the medicinal value of CUR through structural modifications of the molecule and new formulations to increase the oral bioavailability.

To investigate the mechanism of the antifungal effect of CUR against Candida, the sensitivity of CUR to various morphological, iron transporter and oxidative stress mutants of C. albicans was checked and it was observed that only the oxidative stress mutants (ΔCap1 and ΔCaIpf7817) were particularly susceptible to CUR (Figure 4), whereas other mutant strains behaved similarly to the wild-type strain (Supplementary Table S3). A striking feature was the growth inhibitory effects and elevated ROS levels due to CUR, which could be reversed if the natural or synthetic antioxidants were also present in the growth medium (Figures 5 and 6).

The capacity to induce ROS possessed by various antifungals has been reported earlier [11]. For example, azoles such as miconazole, as well as the polyenes amphotericin B and nystatin and polyol macrolides such as niphimycin, induce ROS levels in susceptible fungi [12,14,36,37]. In addition, the benzo-naphthacenequinone antibiotic pradimicin A [38], natural perylenequinonoid pigments [39], the isoprenoid alcohol farnesol [40] and several antifungal peptides/proteins also induce ROS in yeast species. Some of the ROS-inducing antifungals further trigger apoptosis in yeast cells [14,36,38]. An oxidative stress response in yeast is well documented [2729,41]. Yeast cells undergoing apoptosis display several characteristic markers, including the induction of endogenous ROS [42]. With this background, our observation that CUR-induced ROS stimulates the pro-apoptotic regulatory machinery in Candida cells is interesting. We could demonstrate that CUR increased the number of pre-apoptotic cells that could be prevented by the presence of an antioxidant (Figure 8). Raised CaMCA1 levels in the presence of CUR point to caspase-mediated apoptosis in C. albicans (Figure 8C). In our previous study, we have shown that CUR modulates the drug efflux of yeast ABC transporters without affecting the protein levels [43], and here we could demonstrate that CUR exerts its growth inhibitory effects without affecting the transcript levels of genes encoding these transporters (Figure 3C). Hence the effect of CUR on Candida growth is independent of the levels of these transporters (Figure 3).

Interestingly, even at lower concentrations, CUR (37 mg/l) could block the hyphae development in both albicans and non-albicans species of Candida (Figure 9). However, unlike the antifungal effect of CUR, the inhibition of hyphae development could not be reversed by the antioxidants. We found instead that CUR targeted the global repressor TUP1 since CUR could not inhibit hyphae development of Δtup1, which otherwise due to de-repression vigorously makes hyphae. This was further confirmed by Northern-blot experiments where CUR induced an increase in TUP1 transcript levels in wild-type cells (Figure 10). The signalling molecule farnesol, which also causes an increase in ROS levels, exerts its effect via TUP1 [35]. In C. albicans, farnesol is endogenously generated by enzymatic dephosphorylation of farnesyl diphosphate, a precursor for the synthesis of sterols in the sterol biosynthesis pathway [42]. Thus the CUR effect seems to mimic the effect of the quorum sensing molecule farnesol. However, when we checked the effects of CUR on the growth, ROS generation and mycelial development in a knockout of DPP3 that encodes a phosphatase for converting farnesyl pyrophosphate into farnesol, it was observed that CUR continued to inhibit the growth, which could be reversed by the addition of antioxidants in the mutant strain KWN2 (Δdpp3) [35]. The raised ROS levels and the inhibition of hyphae development in DPP3 knockout suggest that CUR only mimics the effects of farnesol phenotypically but is independent of farnesol (Figure 11). Taken together, CUR has dual affects on Candida cells. Its antifungal effect is mediated by the ROS signalling pathway, which brings about an early apoptosis leading to cell death. Independent of the ROS pathway, CUR also inhibits hyphae development by regulating TUP1 levels. Considering the success story of CUR as an anticancer and anti-inflammatory compound, the present study opens up the possibility that CUR can also be exploited as a potential natural antifungal.

Abbreviations

     
  • AA

    ascorbic acid

  •  
  • ABC transporter

    ATP-binding cassette transporter

  •  
  • CaCDR

    Candida albicans Candida drug resistance

  •  
  • CAP1

    Candida albicanse AP-1

  •  
  • CAT1

    catalase 1

  •  
  • cfu

    colony-forming units

  •  
  • CUR

    curcumin

  •  
  • DCFH-DA

    2′,7′-dichlorofluorescein diacetate

  •  
  • DPP3

    diacylglycerol pyrophosphatae phosphatase

  •  
  • FBS

    fetal bovine serum

  •  
  • FI

    fluorescence intensity

  •  
  • FLC

    fluconazole

  •  
  • GRP2

    NADPH-dependent methyl glyoxal reductase

  •  
  • MCA

    metacaspase

  •  
  • MFS

    major facilitator superfamily

  •  
  • MDR

    multidrug resistance

  •  
  • CaMDR

    Candida albicans MDR

  •  
  • PDTC

    pyrrolidinedithiocarbamate

  •  
  • PEG

    poly(ethylene glycol)

  •  
  • PIP

    piperine

  •  
  • PM

    plasma membrane

  •  
  • PS

    phosphatidylserine

  •  
  • ROS

    reactive oxygen species

  •  
  • TUP1

    thymidine uptake 1

  •  
  • YEPD

    yeast extract/peptone/dextrose

We thank Dr Y. Y. Jiang (Department of Phamacology, School of Pharmacy, Second Military University, Shanghai, China) and Dr K. W. Nickerson (School of Biological Sciences, University of Nebraska, Lincoln, NE, U. S. A.) for providing the CaIPF7817 and DPP3 knockout strains respectively. We also thank D. Upadhyay, T. K. Barman and R. Pasrija (Department of Microbiology, Ranbaxy Laboratories Ltd., Gurgaon, India) for assistance with the protocol for in vivo experiments. We are indebted to S. V. Ambudkar (Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, U. S. A.) for his valuable suggestions during the course of this work.

FUNDING

The work was partially supported by the Department of Science and Technology, Indo-DFG [grant number INT/DFG/P-05/2005]; Council of Scientific and Industrial Research, India [grant number 38(1122)/06/EMR-II]; Department of Biotechnology, India [grant numbers BT/PR9100/Med/29/03/2007, BT/PR9563/BRB/10/567/2007, BT/PR11158/BRB/10/640/2008] (Senior Research Fellowship to M.S.); Department of Science and Technology, Indo-Swiss [grant number INT/SWISS/P-31/2009] (all grants to R. P.).

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Supplementary data