Activity of Sanguinarine against Candida albicans Biofilms

ABSTRACT

Candida albicans biofilms show resistance to many clinical antifungal agents and play a considerable contributing role in the process of C. albicans infections. New antifungal agents against C. albicans biofilms are sorely needed. The aim of this study was to evaluate sanguinarine (SAN) for its activity against Candida albicans biofilms and explore the underlying mechanism. The MIC₅₀ of SAN was 3.2 μg/ml, while ≥0.8 μg/ml of SAN could suppress C. albicans biofilms. Further study revealed that ≥0.8 μg/ml of SAN could decrease cellular surface hydrophobicity (CSH) and inhibited hypha formation. Real-time reverse transcription-PCR (RT-PCR) results indicated that the exposure of C. albicans to SAN suppressed the expression of some adhesion- and hypha-specific/essential genes related to the cyclic AMP (cAMP) pathway, including ALS3, HWP1, ECE1, HGC1, and CYR1. Consistently, the endogenous cAMP level of C. albicans was downregulated after SAN treatment, and the addition of cAMP rescued the SAN-induced filamentation defect. In addition, SAN showed relatively low toxicity to human umbilical vein endothelial cells, the 50% inhibitory concentration (IC₅₀) being 7.8 μg/ml. Collectively, the results show that SAN exhibits strong activity against C. albicans biofilms, and the activity was associated with its inhibitory effect on adhesion and hypha formation due to cAMP pathway suppression.

INTRODUCTION

Candida albicans is a common fungal pathogen of humans which may cause life-threatening invasive infections in immunocompromised individuals (1, 2). Available antifungal agents in the clinic are limited, and drug resistance has become a threat (3).

Biofilms are microbial communities consist of cells adhering to medical devices or human organs (4). C. albicans biofilms show resistance to many clinical antifungal agents (5–7) and play a contributing role in the process of C. albicans infections (7). It is urgent to develop new antifungal agents against C. albicans biofilms.

Sanguinarine (SAN; Fig. 1) is a quaternary benzo[c]phenanthridine alkaloid originating from plants of the Papaveraceae family (8, 9). It has extensive pharmacological activities, including antitumor (10), antibacterial (11), anti-inflammatory (12), and antiangiogenesis (13) activities. Nevertheless, its activity against C. albicans biofilms has not yet been intensively investigated. In this study, we revealed the activity of SAN against C. albicans biofilms and found that its antibiofilm activity was associated with the suppression of the cyclic AMP (cAMP) pathway.

FIG 1.

Chemical structure of SAN.

RESULTS

SAN inhibits C. albicans biofilms.

In this study, we first evaluated the antifungal effect of SAN on C. albicans. Antifungal susceptibility testing results indicated that the MIC₅₀ of SAN against C. albicans SC5314 was 3.2 μg/ml, while ≤1.6 μg/ml SAN had little inhibitory effect (Table 1). To make sure that the antifungal effect of SAN is not strain specific, we tested C. albicans ATCC 14053 and two clinical strains (SCZ60053 and SCZ60054). SAN exhibited antifungal effect on all the strains tested (Table 1). Then we conducted a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and determined the inhibitory effect of SAN against C. albicans biofilm formation. Adding SAN to C. albicans cells after 90 min of adhesion inhibited biofilm formation in a dose-dependent manner (Fig. 2A). More specifically, 0.4 μg/ml of SAN inhibited biofilm formation by 26.9% (P < 0.001), and the inhibitory effect increased as the SAN concentration increased. SAN at 1.6 μg/ml inhibited biofilm formation by 72.9% (P < 0.0001). Moreover, SAN exhibited activity against mature biofilms (Fig. 2B). At 0.8 μg/ml, SAN destroyed mature biofilms by 23.3% (P < 0.001). As SAN concentration increased, the effect was more obvious. SAN at 3.2 μg/ml destroyed mature biofilms by 68.3% (P < 0.0001). In contrast, as much as 1,024 μg/ml of fluconazole could inhibit biofilm formation only by about 60% (Fig. 2A) or destroy mature biofilms by 29.9% (Fig. 2B).

TABLE 1.

MICs of SAN and fluconazole against C. albicans strains^a

Strains	SAN		Fluconazole
	MIC80	MIC50	MIC80	MIC50
SC5314	3.2	3.2	0.25	0.125
ATCC 14053	3.2	1.6	0.5	0.25
SCZ60053	3.2	1.6	>64	0.25
SCZ60054	3.2	1.6	>64	0.125

^aValues are in micrograms per milliliter.

FIG 2.

SAN inhibits C. albicans biofilm formation in vitro. Biofilm formation was evaluated using an XTT reduction assay, and the results are presented as the percent SAN- or fluconazole-treated biofilm relative to control biofilm formed without treatment. The results represent means ± standard deviations for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way analysis of variance [ANOVA]). (A) Effects of different concentrations of SAN or fluconazole on biofilm formation. (B) Effects of different concentrations of SAN or fluconazole on mature biofilms. (C) Effects of different concentrations of SAN on biofilm formation, shown in SEM images. Images in the dotted boxes are enlarged in the rightmost column.

The antibiofilm effect of SAN was confirmed by scanning electron microscopy (SEM). The hyphae in C. albicans biofilms were inhibited by SAN in a dose-dependent manner (Fig. 2C), and at 3.2 μg/ml of SAN, criss-crossing true hyphae could not be observed (Fig. 2C, panels j to l).

SAN decreases CSH of C. albicans.

Since adhesion and morphological transition are two important stages for C. albicans biofilm formation (14) and there is a positive correlation between cellular surface hydrophobicity (CSH) and adhesion (15–17), we investigated the influence of SAN on CSH of C. albicans. The normal relative CSH of C. albicans was about 89.9% (Fig. 3). SAN at 0.8 μg/ml significantly decreased the CSH of C. albicans, to approximately 42.6% (P < 0.001). Moreover, SAN decreased the CSH in a dose-dependent manner. The CSH of C. albicans approached 0 with SAN treatment at 3.2 μg/ml.

FIG 3.

SAN decreases the relative CSH of C. albicans. The results represent means ± standard deviations for three independent experiments; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA).

SAN inhibits hypha formation of C. albicans.

The effect of SAN on C. albicans hypha formation was further evaluated by incubating C. albicans in several hypha-inducing media, including RPMI 1640, Spider, and Lee media. C. albicans could form hyphae in all the hypha-inducing media, while SAN inhibited hypha formation in all the media tested (Fig. 4). More specifically, hypha formation was completely inhibited by 0.8 μg/ml of SAN in Spider medium and all C. albicans cells were maintained as yeasts. In RPMI 1640 and Lee media, SAN inhibited hypha formation in a dose-dependent manner.

FIG 4.

Effects of different concentrations of SAN on hypha formation. Exponentially growing C. albicans SC5314 cells were transferred to hypha-inducing liquid media. The cellular morphology was photographed after incubation at 37°C for 3.5 h. Bars, 100 μm.

Exposure of C. albicans to SAN suppresses the expression of genes related to the cAMP pathway.

Since SAN could inhibit adhesion and hypha formation, we detected the expression of some well-known genes (18, 19) involved in adhesion and filamentation (Fig. 5A). A total of 1.6 μg/ml of SAN was used in this assay because SAN at this concentration had a significant antibiofilm effect but little fungicidal effect. Twenty-six genes were investigated. Our real-time reverse transcription-PCR (RT-PCR) results indicated that ALS3, HWP1, ECE1, HGC1, and CYR1 were downregulated after SAN treatment (Fig. 5A). Interestingly, these downregulated genes are all cAMP pathway related (19).

FIG 5.

The effect of SAN is associated with suppression of the cAMP pathway. (A) Real-time RT-PCR results for some important adhesion/filamentation-related genes. Gene expression fold changes are relative to results of the control group (C. albicans SC5314 without SAN treatment). 18S rRNA was used to normalize the expression data. Data are means ± standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test). (B) Endogenous cAMP levels of C. albicans. Intracellular cAMP levels of C. albicans in the group treated with 1.6 μg/ml of SAN and the control group were determined. The results represent means ± standard deviations from three independent experiments. *, P < 0.05 (unpaired t test). (C) Exogenous cAMP reverted the morphological transition defect of C. albicans caused by SAN. The cellular morphology was photographed after incubation in RPMI 1640 medium at 37°C for 3.5 h. Bars, 100 μm.

SAN downregulates the level of endogenous cAMP, and the addition of cAMP rescues the morphogenesis defect caused by SAN.

Since a series of cAMP pathway-related genes were downregulated after SAN treatment, we hypothesized that the antibiofilm effect of SAN was related to the suppression of the cAMP pathway. The endogenous cAMP level of C. albicans was determined. As expected, the endogenous cAMP level of C. albicans treated with 1.6 μg/ml of SAN was significantly lower than that in control group (P < 0.05 [Fig. 5B]).

We further investigated the effect of exogenous cAMP on the SAN-induced morphological transition defect. RPMI 1640 medium was chosen for the cAMP experiment because this medium is the most widely used in biofilm experiments (18, 20, 21). A total of 1.6 μg/ml of SAN was used because SAN at this concentration could inhibit hypha formation obviously. The results showed that exogenous cAMP rescued the morphogenesis defect caused by SAN. Specifically, the addition of 3.2 mg/ml of cAMP restored the hypha formation of C. albicans with 1.6 μg/ml of SAN in RPMI 1640 medium (Fig. 5C).

SAN exhibits relatively low toxicity to mammalian cells.

We assessed the toxicity of SAN using human umbilical vein endothelial cells (HUVECs). SAN at 1.6 μg/ml had no observable influence on viability of cells. SAN at 12.8 μg/ml began to exhibit obvious toxicity toward the mammalian cells (Fig. 6). The 50% inhibitory concentration (IC₅₀) of SAN on HUVECs was 7.8 μg/ml. Collectively, the results demonstrated that SAN had a weaker suppressive effect on mammalian cells than on C. albicans cells.

FIG 6.

Toxicity of SAN to HUVECs. The results represent means ± standard deviations from three independent experiments. *, P < 0.05; ****, P < 0.0001 (one-way ANOVA).

DISCUSSION

C. albicans biofilms have become a threat to successful antifungal treatment, and antibiofilm agents are urgently needed (6). In this study, we revealed a significant effect of SAN against C. albicans biofilms, including biofilm formation and the maintenance of mature biofilms. We also found that SAN could decrease CSH and suppress the yeast-to-hypha morphological transition. The results of real-time RT-PCR indicated that some important adhesion-related genes and filamentation genes were differentially expressed after exposure to SAN. We further revealed that the effect of SAN was related to the cAMP pathway, and exogenous cAMP could rescue the hyphal morphogenesis defect caused by SAN.

SAN has a strong activity against C. albicans biofilms. More importantly, SAN could not only inhibit the formation of biofilms but also destroyed the maintenance of mature biofilms. At 1.6 μg/ml ([1/2]× MIC₅₀), SAN reduced biofilm formation by more than 70% and destroyed the maintenance of mature biofilms by about 45%. The effect of SAN was outstanding compared with those of various other antifungal agents. Fluconazole could not inhibit biofilm formation in a dose-dependent manner and could hardly affect mature biofilms. Similarly, Vila et al. (22) reported that amphotericin B at 1× MIC inhibited the development of biofilms but could not affect the maintenance of mature biofilms. In additions, our lab has reported the antibiofilm activities of pterostilbene and tetrandrine previously (18, 20). About 7.7 μg/ml of pterostilbene or 31.1 μg/ml of tetrandrine is needed to reduce biofilm formation by about 60% (18, 20). Our findings in this study indicate that the antibiofilm activity of SAN is significantly stronger than those of pterostilbene and tetrandrine.

Our data also indicated that SAN may inhibit biofilm formation by preventing adhesion (with CSH as the indicator [17]) and morphological transition. There are three known stages for biofilm formation: adhesion to biomaterial surfaces, growth to form an anchoring layer, and morphological transition to form a complex three-dimensional structure (14, 23–25). Our data indicated that 1.6 μg/ml of SAN obviously inhibited biofilm formation, significantly decreased the CSH, and apparently inhibited the yeast-to-hypha morphological transition, while SAN could not affect the growth of C. albicans obviously at this concentration. Therefore, it seems that the antibiofilm activity of SAN can be attributed to its antiadhesion and anti-morphological transition activities.

To clarify the antibiofilm mechanism of SAN, real-time RT-PCR was performed in this study. Some important adhesion- and hypha-related genes were downregulated after SAN treatment. More specifically, ALS3 is an ALS family gene that plays an essential role in the adherence stage of C. albicans (26, 27). HWP1 is a unique adhesion gene expressed on the hyphal surface, and biofilms lacking HWP1 were prone to detach from the abiotic substrate (28). ECE1 is a hypha-specific gene related to the extent of hyphal cell elongation (29). HGC1 functions in the maintenance of hyphal growth (30). CYR1 encodes adenylyl cyclase in the cAMP pathway and is essential for hypha formation (31). The downregulation of these genes may contribute to the antibiofilm effect of SAN.

Interestingly, the downregulated genes, including ECE1, ALS3, HWP1, and HGC1, are all regulated by the cAMP pathway (32). Moreover, another downregulated gene, CYR1, is an essential gene functioning in the cAMP pathway (19, 33). Based on these findings, we speculate that the antibiofilm effect of SAN might be related to the suppression of cAMP pathway. As expected, the endogenous cAMP level was decreased after SAN treatment and exogenous cAMP rescued the morphogenesis defect caused by SAN. SAN may inhibit adhesion and filamentous growth of C. albicans by suppressing the cAMP pathway.

SAN exhibited relatively low toxicity to mammalian cells in this study: 1.6 μg/ml of SAN had no observable influence on viability of HUVECs, and the IC₅₀ of SAN was 7.8 μg/ml. Watamoto et al. (34) also tested the toxicity of SAN to mammalian cells and obtained an IC₅₀ of 0.73 μM (about 0.27 μg/ml). The difference may originate from the different compounds and different cells used. We used sanguinarine with a 99% purity level and HUVECs, while Watamoto et al. used sanguinarine chloride hydrate with a 98% purity level and human gingival fibroblasts. Based on our findings, SAN is relatively safe to mammalian cells. Nevertheless, further study of the structure-activity relationship is needed to reveal SAN analogues with higher antibiofilm activity and lower toxicity to mammalian cells as candidates for drug development.

In summary, SAN exhibits a strong antibiofilm effect against C. albicans, which is associated with the downregulation of the cAMP pathway. SAN may act as a lead compound for drug development to treat life-threatening biofilm-related infections caused by C. albicans. Nevertheless, further translational research is required to determine whether the antibiofilm effect of SAN is applicable in clinical settings.

MATERIALS AND METHODS

Strains, culture, and agents.

C. albicans SC5314 is a widely used strain in C. albicans research (20, 35) and was used in this study. The strain can form normal biofilms and is susceptible to fluconazole (20, 35). Other clinical C. albicans strains used in this study include ATCC 14053, SCZ60053, and SCZ60054. The strains were routinely grown in YPD liquid medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30°C in a shaking incubator (20). Human umbilical vein endothelial cells (HUVECs) were incubated in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and were used to evaluate the toxicity of SAN (36–39). SAN was bought from Vita Chemical Reagent Co., and the purity level was 99%. For all the experiments, 12.8 mg/ml of SAN in dimethyl sulfoxide (DMSO; Sigma-Aldrich) was used as a stock and a 10-fold dilution of the stock was made in DMSO before addition to the culture suspensions to obtain the required SAN concentrations. Other media include RPMI 1640 medium (40), Spider medium (33), and Lee medium (41). To prepare the media mentioned above, mineral salts were purchased from Sangon Biotech (China); amino acids were purchased from Sigma-Aldrich; RPMI 1640, DMEM, and FBS were purchased from Gibco (USA); and other ingredients were purchased from BD (USA).

Antifungal susceptibility testing.

Antifungal susceptibility testing was performed according to the broth microdilution protocol of the Clinical and Laboratory Standards Institute (M27-A3), with a few modifications (20, 40, 42, 43). Briefly, the initial concentration of fungal suspension in RPMI 1640 medium was 5 × 10³ CFU/ml, and the final concentration of SAN ranged from 0.1 to 25.6 μg/ml. The plates were incubated at 35°C for 24 h. The growth inhibition was determined by the optical densities at 600 nm (OD₆₀₀), from which the background optical densities were already subtracted. Fluconazole served as a positive control. Each drug was tested in triplicate. The MIC₅₀s and MIC₈₀s were determined.

In vitro biofilm formation assay.

The in vitro biofilm formation assay was carried out as described previously (6, 18, 20). In brief, 1 × 10⁶ CFU/ml of cells in RPMI 1640 medium were added to a 96-well tissue culture plate for 90 min of adhesion at 37°C. After adhesion, the medium was aspirated, nonadherent cells were removed, and fresh RPMI 1640 was added. The plate was further incubated at 37°C for 24 h until formation of mature biofilms. The formed biofilms were calculated with a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, which is a kind of semiquantitative measurement (44). To detect the effect of SAN on the formation of biofilms, different concentrations of SAN were added to fresh RPMI 1640 after 90 min of adhesion. To detect the effect of SAN on mature biofilms, C. albicans biofilms were formed at 37°C for 24 h as described above. The biofilm supernatant was then aspirated, and fresh RPMI 1640 medium containing different concentrations of SAN was added. The plates were incubated at 37°C for another 24 h to observe the antibiofilm effect of SAN.

SEM.

For scanning electron microscopy (SEM), glass disks coated with poly-l-lysine hydrobromide were used to develop biofilms. Biofilms were washed and placed in a fixative consisting of 2% (vol/vol) glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.2) for 2 h. The samples were rinsed twice in cacodylate buffer, garnished with 1% osmic acid for 2 h, dehydrated in an ascending ethanol series, treated with hexamethyl-disilazane, and dried overnight. The specimens were coated with gold and observed through a Philips XL-30 SEM (Philips) in high-vacuum mode (20, 45).

CSH assay.

Cellular surface hydrophobicity (CSH) was measured by a water-hydrocarbon two-phase assay as described previously, with a few modifications (20, 44, 46). In brief, the formed C. albicans biofilms were removed from the culture plate surface to obtain a cell suspension (OD₆₀₀ = 1.0 in phosphate-buffered saline [PBS]). Then 1.2 ml of the suspension was pipetted into a clean glass tube and overlaid with 0.3 ml pf octane. The mixture was vortexed for 3 min and then stood at room temperature for another 15 min for phase separation. The OD₆₀₀ of the aqueous phase was determined, and the OD₆₀₀ for the group without the octane overlay was used as the control. Three repeats were performed for each group. Relative hydrophobicity was calculated as follows: [(OD₆₀₀ of control − OD₆₀₀ after octane overlay)/OD₆₀₀ of control] × 100.

Real-time RT-PCR.

Real-time reverse transcription-PCR (RT-PCR) was conducted as described previously (20, 23). Briefly, C. albicans biofilms were formed in 150-mm by 25-mm polystyrene cell culture dishes. C. albicans cells were collected and washed, and total RNA was isolated by a fungal RNAout kit (60305-50; TIANZ). cDNA was obtained through a reverse transcription reaction performed with a reverse transcription kit (RR037A; TaKaRa Biotechnology). Real-time PCR was performed with a 7500 real-time PCR system (Applied Biosystems); primers are shown in Table S1 in the supplemental material. SYBR green I (RR420A; TaKaRa) was used to monitor the amplified products. The expression of each gene was normalized to that of 18S rRNA. The relative expression of each target gene was calibrated against the corresponding expression by untreated C. albicans SC5314 (expression = 1), which served as the control. Triplicate independent experiments were conducted to generate a mean value.

Endogenous cAMP assay.

C. albicans cells were collected using the same protocol as described for real-time RT-PCR. The endogenous cAMP level was determined using a cAMP complete enzyme-linked immunosorbent assay (ELISA) kit (ab133051; Abcam) according to the manufacturer's instructions.

MTT viability assay.

HUVECs were used to evaluate the toxicity of SAN (37–39). A total of 1 × 10⁵ cells/ml of HUVECs in medium were seeded in 96-well tissue culture plates and incubated at 37°C for 24 h in the presence of 5% CO₂. After incubation, the supernatant was removed and fresh media with different concentrations of SAN were added. The plates were incubated for another 24 h. Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma) as described previously (36, 47). Briefly, the supernatant was removed and 100 μl of MTT (0.5 mg/ml in PBS) was added to each well and incubated at 37°C for 4 h. The supernatant was removed again and 100 μl of DMSO was added for 15 min. Cell viability was assessed by measuring absorbance at 570 nm. Cells incubated in DMEM without SAN treatment were set as the standard for 100% viability.