The standard C. albicans strains DAY185, ATCC10231, ATCC18804, and ATCC24433 used in this study were obtained from the Korean Culture Center of Microorganisms (http://www.kccm.or.kr/). Streaking and subculturing of C. albicans strains were performed using potato dextrose agar (PDA) or potato dextrose broth (PDB), unless otherwise specified. C. albicans strain was preserved at −80°C in 1 ml of PDB supplemented with 30% glycerol stock, and when needed, streaked on PDA plates. Plates were incubated for 48 h at 37°C, and a fresh single colony was then inoculated into 25 ml of PDB and cultured overnight at 37°C. The strain S. aureus 6538 was maintained and cultured in LB medium. All 13 compounds tested, namely, alizarin, alizarin red, anthraflavic acid, anthraquinone, chrysazin, (+)-catechin, (+)-catechin hydrate, emodin, 1-hydroxyanthra-9,10-quinone, hydroquinone, purpurin, pyrocatechol, and quinalizarin were purchased from Sigma-Aldrich (St. Louis, USA) and dissolved in dimethyl sulfoxide (DMSO), which did not exceed 0.1% (vol/vol) in any experiment. To determine cell growths, turbidities were measured at 620 nm using a spectrophotometer (UV-160, Shimadzu, Japan). Minimum inhibitory concentrations (MICs) were determined using the Clinical Laboratory Standards Institute (CLSI) broth dilution method with slight modification (Alastruey-Izquierdo et al., 2015), using 96-well polystyrene plates (SPL Life Sciences, Korea). C. albicans cells were cultured overnight in PDB medium and diluted to reach a final concentration of 10⁵ CFU/mL and added to the wells in the presence of varying concentrations (w/v) of tested compounds at 24 h at 37°C. MIC was defined as the lowest concentration that inhibited microbial growth by at least 80%, as assessed by spectrophotometry (620 nm) and colony counting. MICs of tested compounds are expressed as percentages (v/v or w/v).

Candida biofilms were prepared on 96-well polystyrene plates, as previously reported (Lee et al., 2011). Briefly, overnight cultures of C. albicans strains were inoculated into PDB (total volume 300 μl) at an initial turbidity of 0.1 at 600 nm and cultured with or without test compounds at varying concentrations for 24 h without shaking at 37°C. To perform biofilm inhibition assay in mixed cultures, overnight cultures of C. albicans and S. aureus strains were equally inoculated into medium containing PDB and LB (final volume 300 μl) at an initial turbidity of 0.1 at 600 nm and cultured as mentioned above.

To investigate the effects of the anthraquinones alizarin and chrysazin on biofilm disruption, C. albicans biofilms were prepared on 96-well polystyrene plates for 24 h at 37°C, as described above. Briefly, wells were washed twice with PBS and fresh PDB medium (300 μl) containing different concentrations of compounds were added to plates, which were then further incubated for 24 h at 37°C. Biofilm formation was quantified after washing three times with H₂O (to remove all non-adherent cells), staining with crystal violet for 20 min, rinsing three times with H₂O, and extracting the crystal violet with 95% ethanol. Absorbance was measured at 570 nm, and results are presented as the averages of at least six replicates.

A colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] reduction assay was performed using established procedures (Ramage et al., 2001; Nett et al., 2011). Briefly, overnight cultures of C. albicans strains were inoculated into PDB (total volume 300 μl) at an initial turbidity of 0.1 at 600 nm and cultured with or without alizarin or chrysazin at different concentrations for 24 h without shaking at 37°C. A XTT reduction kit (Sigma-Aldrich, St. Louis, USA) was used to measure the metabolic activities of biofilm cells. XTT and menadione solutions were mixed at 20:1 (v/v) immediately prior to the assay. PBS was then added to XTT-menadione solution (3.76:1 v/v) and 200 μl of this mix was added to each well containing pre-washed biofilms, and incubated in the dark for 3 h at 37°C. The colored supernatant (100 μl) so obtained was transferred to new microtiter plates, and optical densities were measured at 450 nm. Similarly, planktonic cell viability was measured by using culture supernatants.

Overnight cultures of C. albicans DAY 185 were inoculated into the hyphae-inducing media RPMI-1640 medium with or without alizarin or chrysazin (both at 2 μg/ml) for 24 h with shaking at 37°C. Aliquots of fungal cells were harvested at different times (0, 6, or 24 h) and visualized under bright field using the iRiS™ Digital Cell Imaging System (Logos Bio Systems, Korea).

C. albicans cells were cultured in 96-well polystyrene plates (SPL Life Sciences, Korea) without shaking in the absence or presence of alizarin or chrysazin. Planktonic cells were then removed by washing with PBS buffer three times. C. albicans cells were stained with carboxyfluorescein diacetate succinimidyl ester (a minimally fluorescent lipophile; Catalog #: C34554, Invitrogen, Molecular Probes, Inc, Eugene, USA; Weston and Parish, 1990), which becomes highly fluorescent when it loses its acetyl groups due to the action of esterases in cells. Biofilms at the bottom of plate was visualized using an (a 488 nm) Ar laser (emission wavelength 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo) equipped with a 20 × objective (Kim et al., 2012). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were examined.

SEM was used to observe hyphal formation, as previously described (Lee et al., 2014). Briefly, a nylon membrane was cut into 0.5 × 0.5 cm pieces and one piece was placed per well in 96-well plates containing 200 μL cells/well of turbidity 0.05 at 600 nm. Cells were incubated in the presence or absence (untreated control) of alizarin or chrysazin at 37°C for 24 h without shaking. Cells were then fixed with glutaraldehyde (2.5%) and formaldehyde (2%) for 24 h, and post fixed in osmium tetroxide and dehydrated in a series of ethanol solutions (50, 70, 80, 90, 95, and 100%), and isoamyl acetate. After critical-point drying, cells fixed onto nylon membranes were examined under a S-4200 scanning electron microscope (Hitachi, Japan) at a voltage of 15 kV and magnifications ranging from × 2,000 to × 10,000.

For transcriptional analysis, C. albicans was inoculated into 25 ml of PDB broth in 250 ml shake flasks at a starting OD₆₀₀ of 0.1, and then cultured at 37°C for 4 h with agitation (250 rpm) in the presence or absence of alizarin or chrysazin (2 μg/ml). RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a hot acidic phenol method (Amin-ul Mannan et al., 2009) and further proceed to clean up this RNA with Qiagen RNeasy mini Kit (Valencia, CA, USA). qRT-PCR was used to determine the transcription levels of hypha- and biofilm-related genes [HYR1, EFG1, ECE1, ECE2, ALS1, ALS3 (or called HWP1), EED1, and RBT1] in C. albicans treated or not with alizarin or chrysazin. Gene specific primers were used and RDN18 was used as housekeeping controls (Supplementary Table 1). The qRT-PCR method used has been previously described (Lee et al., 2011). qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures.

To investigate the effects of alizarin or chrysazin or purpurin on the virulence of C. albicans, the nematode C. elegans wild type strain N2 Bristol CF512 fer-15(b26); fem-1(hc17) (Murphy et al., 2003; Oh et al., 2012) with loss of sex-determining protein Fem-1 and age determining protein Fer-15 was infected with C. albicans as previously described (Manoharan et al., 2017a). Briefly, a freshly prepared overnight C. albicans culture (100 μl) was spread onto a lawn on PDA plates and incubated for 48 h at 37°C. Synchronized adult were then allowed to feed on the C. albicans yeast lawn for 4 h at 25°C when worms were collected and washed three times with sterile M9 buffer. Approximately 10 worms were then pipetted into single wells of 96-well plates containing PDB medium and treated with solutions (300 μl) of investigated compounds at final concentrations ranging from 0.2 to 2 μg/ml. Controls were treated with medium alone. For toxicity assays, 10 non-infected worms were pipetted into single wells of a 96-well dish containing M9 buffer and solutions of the compounds (300 μl) were added to final concentrations of 1 or 2 mg/ml. Plates were then incubated at 25°C for 4 days with gentle shaking. Three independent experiments were conducted in triplicate. Results are expressed as percentages of alive or dead worms by gently touching them with a platinum wire after 4 days of incubation, and results were obtained using an iRiS™ Digital Cell Imaging System (Logos Bio Systems, Korea).

In all cases, at least two independent experiments were conducted, and results are expressed as means ± standard deviations. The student's t-test was used to determine the significances of differences between treated and non-treated samples. Statistical significance was accepted for p < 0.05, and in the figures significant changes are indicated by asterisks.

The antibiofilm efficacies of the 13 compounds (Figure 1A) tested were examined using C. albicans on 96-well polystyrene plates. Of these compounds, the anthraquinones alizarin, chrysazin, purpurin, 1-hydroxyanthra-9,10-quinone, emodin, and quinalizarin were found to be effective at 10 μg/ml against C. albicans biofilms without affecting planktonic cell growth (Figure 1B). Nevertheless, anthraquinone, pyrocatechol, and alizarin red showed significant biofilm inhibition at 50 μg/ml (Figure 1B). Further experimentation showed that chrysazin, purpurin, and alizarin significantly reduced biofilm formation by 50, 57, and 82%, respectively, even at 0.5 μg/ml (Figure 1C). Further increases in the concentrations of alizarin, chrysazin, and fluconazole significantly exacerbated biofilm disruption (Figure 2A). In addition, alizarin and chrysazin at 2 μg/ml dramatically reduced biofilm formation by three other C. albicans strains (ATCC10231, ATCC18804, ATCC24433) by ≥90%. While fluconazole significantly reduced biofilm formation by C. albicans strains ATCC10231 and ATCC24433 by 43 and 72%, respectively at 2 μg/ml (Figure 2B). Our data suggest that alizarin and chrysazin are more effective to inhibit biofilm formation by several C. albicans strains than commercial drug fluconazole. Interestingly, alizarin, chrysazin, and purpurin had significantly reduced mixed biofilm formation by C. albicans and S. aureus at 2 and 10 μg/ml, while fluconazole had lower activity on mixed biofilms at 10 μg/ml when compared to anthraquinone derivatives (Supplementary Figure 1). Since purpurin has been reported as antibiofilm and antihyphal agent against C. albicans (Tsang et al., 2012), our present study was designed and focused only on alizarin and other anthraquinone related compounds for their antihyphal activities.

The effects of alizarin and chrysazin on C. albicans biofilms and planktonic cells were quantified using XTT reduction assay and viabilities were calculated by expressing metabolic activities as percentages of non-treated controls. C. albicans metabolic activity was significantly reduced (by 80%) by alizarin up to 2 μg/ml. However, chrysazin reduced the metabolic activity of C. albicans biofilms by 66% at 2 μg/ml (Figure 3), and alizarin at 10 μg/ml completely removed biofilms (>98%), while it slightly reduced (25%) metabolic activity on planktonic cells.

The antimicrobial activities of alizarin and chrysazin were investigated by measuring minimum inhibitory concentrations (MIC), which for both compounds were >2,000 μg/ml against C. albicans. Notably, this MIC was 1,000-times higher than the concentration (2 μg/ml) required for antibiofilm activity for both compounds. The MIC of all quinone and anthraquinone derivatives are given in Table 1. C. albicans DAY185 was found to be resistant to all tested compounds. For example, anthraflavic acid had showed a MIC up to 500 μg/ml, several compounds exhibited MIC ranging from 1,000 to 5,000 μg/ml. These results confirm that biofilm formation by C. albicans was effectively reduced by the antibiofilm activities of alizarin and chrysazin and not by their antifungal activities. These results show that alizarin and chrysazin have fungistatic rather than fungicidal effects on C. albicans.

Confocal laser scanning microscopy (CLSM) images showed C. albicans formed dense biofilms in non-treated control samples, but that in the presence alizarin at 0.2 and 2 μg/ml biofilm cellular densities and thicknesses were reduced. While no reduction was observed at a chrysazin concentration of 0.2 μg/ml, major reduction was observed at 2 μg/ml (Figure 4). Scanning electron microscopy (SEM) showed in addition to reducing biofilm metabolic activity, alizarin also inhibited yeast to hyphal transition. As shown in Figure 5, nontreated control biofilms consisted of mixtures of pseudohyphae and hyphae, and few yeast cells. In contrast, alizarin and chrysazin reduces the length of existing hyphae of C. albicans and increased yeast form cells as compared to the untreated control (Figure 5). In addition, alizarin and chrysazin both strongly inhibited biofilm development on polystyrene surfaces (Figures 1, 4) and nylon membrane (Figure 5) as well.

Hyphae inducing RPMI medium was used to examine hyphal inhibition by alizarin and chrysazin. Untreated controls showed massive hyphal growth after 6 h, but in the presence of 2 μg/ml alizarin or chrysazin hyphal growth was not observed. When cultured in RPMI containing alizarin or chrysazin at 2 μg/ml for 24 h, fungal cells grew by budding, whereas controls large hyphal cells were observed (Figure 6) Microscopic observations of alizarin or chrysazin treated fungal cells revealed that in addition to reducing metabolic activity in biofilms, both compounds reduced hyphal lengths and inhibited yeast to hyphae transition.

Transcriptional levels of the hypha-specific and biofilm related genes in C. albicans were quantified by qRT-PCR. Alizarin at 2 μg/ml significantly downregulated the expression of the hypha specific genes ALS3 (2.4-fold), ECE1 (3.7-fold), ECE2 (6.3-fold), and RBT1 (5.8-fold) when compared to their respective controls (Figure 7). Similarly, the expression of ECE1, ECE2, and RBT1 were significantly downregulated by 2-, 2.3-, and 2-fold, respectively after chrysazin (2 μg/ml) treatment. Nevertheless, HYR1 (hypha-specific gene) and EED1 (adhesive related gene) were not affected after alizarin treatment. Interestingly, ALS1 which involved in adhesion and biofilm formation was found to be upregulated by alizarin treatment. Similarly, the transcriptional level of hyphae regulatory gene EFG1 was increased after alizarin treatment. Taken together, qRT-PCR results showed that alizarin significantly altered the expression of some hypha-specific genes and adhesive related genes.

The antibiofilm and antihyphal effects of alizarin and chrysazin were examined in C. elegans infected with C. albicans. Microscopic observations of infected nematodes revealed that C. albicans infection caused 95% fatality in 4 days. However, more than 60% of nematodes survived 4 days in the presence of alizarin (2 μg/ml), >50% survived 4 days in the presence of chrysazin, >60% survived 4 days in the presence of purpurin and <50% survived 4 days in the presence of fluconazole (a commercial antifungal agent; Figure 8A). To examine the toxicities of alizarin and chrysazin, non-infected worms were treated with different concentrations for 4 days. It was found alizarin at concentrations of 1 mg/ml did not affect nematode viability or survival (Figure 8B). However, at this concentration, chrysazin reduced nematodes survival by >60% and purpurin reduced 35% survival rate, whereas only <5% nematodes survived treatment with fluconazole at 1 mg/ml for 4 days. These results show alizarin was as effective as fluconazole at promoting the survival of infected nematodes, but that it was substantially less toxic.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.