The Glucocorticoid PYED-1 Disrupts Mature Biofilms of Candida spp. and Inhibits Hyphal Development in Candida albicans

Invasive Candida infections have become a global public health problem due to the increase of Candida species resistant against antifungal therapeutics. The glucocorticoid PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1) has antimicrobial activity against various bacterial taxa. Consequently, it might be considered for the treatment of Candida infections. The antifungal activity of PYED-1 was evaluated against several fungal strains that were representative of the five species that causes the majority of Candida infections—namely, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei. PYED-1 exhibited a weak antifungal activity and a fungistatic effect on all five Candida species. On the other hand, PYED-1 exhibited a good anti-biofilm activity, and was able to eradicate the preformed biofilms of all Candida species analyzed. Moreover, PYED-1 inhibited germ tube and hyphae formation of C. albicans and reduced adhesion of C. albicans to abiotic surfaces by up to 30%.


Introduction
Candidemia and invasive candidiasis are severe healthcare-associated infections with a high mortality rate and limited therapeutic options [1,2]. Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei are the five species that cause approximately 90% of all infections [3]. C. albicans is the most prevalent cause of invasive fungal infections. The therapeutic failures of C. albicans infections are quite high [4], although the levels of antifungal resistance reported for this organism are very low [5]. Recent studies reported a growing proportion of non-albicans Candida infections [6]. Patients with a non-albicans Candida infection have a less effective response to antifungal therapies and, as a consequence, a high degree of mortality [4]. Candida spp. can form biofilm on biotic and abiotic surfaces, becoming less susceptible to antifungal drugs. Biofilm formation is highly associated with persistent candidemia [7].
The increasing resistance of fungal species to antifungal drugs has led to a need for the development of novel therapeutic agents with a broad spectrum of activity, along with low toxicity and high efficacy against all Candida species.
As part of our interest in the synthesis of compounds endowed with pharmacological activity [8][9][10][11][12], we recently developed a novel synthetic route aimed at the preparation of the corticosteroid drug deflazacort (DFZ, Figure 1) in order to explore its antimicrobial activity. the corticosteroid drug deflazacort (DFZ, Figure 1) in order to explore its antimicrobial activity. In this context, while DFZ showed no activity, one of its synthetic precursors, the heterocyclic corticosteroid PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1), showed significant antimicrobial, antibiofilm and anti-virulence activities against Gram-positive and Gram-negative bacteria without showing cytotoxicity at concentrations up to 128 µg/mL [13][14][15][16]; nevertheless, its ability to interfere with fungal pathogens has not yet been investigated. Herein we describe the evaluation of the corticosteroid PYED-1 as an inhibitor of the growth and biofilm formation in five species of Candida, namely, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

Antifungal Susceptibility to Glucocorticoid PYED-1 of Candida Species
The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of PYED-1 were determined for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei clinical isolates. The drug fluconazole was used as a positive control. Results in Table 1 show that PYED-1 had anticandidal activity on all clinical isolates of all Candida spp. assayed.  In this context, while DFZ showed no activity, one of its synthetic precursors, the heterocyclic corticosteroid PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1), showed significant antimicrobial, antibiofilm and anti-virulence activities against Grampositive and Gram-negative bacteria without showing cytotoxicity at concentrations up to 128 µg/mL [13][14][15][16]; nevertheless, its ability to interfere with fungal pathogens has not yet been investigated. Herein we describe the evaluation of the corticosteroid PYED-1 as an inhibitor of the growth and biofilm formation in five species of Candida, namely, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

Antifungal Susceptibility to Glucocorticoid PYED-1 of Candida Species
The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of PYED-1 were determined for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei clinical isolates. The drug fluconazole was used as a positive control. Results in Table 1 show that PYED-1 had anticandidal activity on all clinical isolates of all Candida spp. assayed. To test whether PYED-1 can synergize with available antifungal drugs, checkerboard assays were performed. Fluconazole or posaconazole had neither synergistic nor antagonistic interactions with PYED-1 (data not shown), indicating that PYED-1 co-treatment cannot impair or attenuate the efficacy of these antifungal drugs.

Effect of PYED-1 on Preformed Candida spp. Biofilm
The formation of fungal biofilm on biotic and abiotic surfaces is a major virulence factor in Candida spp. It causes a reduction in the penetration and, therefore, in the efficacy of antifungal drugs. It contributes to the appearance of drug resistance and recurrent fungal infections in the clinic [17]. Therefore, it is of interest to identify novel molecules with antibiofilm activity. The antibiofilm activity of PYED-1 was evaluated against preformed biofilms of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis strains by XTT (2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction and crystal violet (CV) assays. As shown in Figure 2, results varied according to the Candida species.
In the eradication biofilm assay, exposure of preformed C. glabrata biofilms to 128, 64 and 32 µg/mL of PYED-1 reduced the cell viability by 75-92% ( Figure 2A) and the biofilm biomass by 30-70% ( Figure 2B), as compared to untreated controls. PYED-1 at 64 µg/mL was able to reduce the number of viable C. tropicalis to over 80% (Figure 2A), while biofilm biomass was diminished by 50% ( Figure 2B). In contrast, PYED-1 displayed a significant reduction in the number of viable cells of preformed biofilms of C. albicans and C. parapsilosis when compared to untreated controls, but only marginally reduced the biofilm biomass ( Figure 2). Candida biofilms are widely considered to be a key determinant of the high mortality rate attributed to candidiasis [7]. Candida can form biofilms on biomedical implants that are difficult to eradicate by clinical drugs such as amphotericin B, fluconazole, flucytosine and itraconazole. They are also responsible for the high resistance to antifungal drugs [18]. Antifungal resistance due to biofilm formation is frequently reported in clinical settings, and it counteracts the achievement of effective treatment strategies [19]. Antibiofilm agents represent a novel approach to reducing antifungal resistance, destroying preformed biofilm and making the persistent cells susceptible to antifungal drugs [20]. Thus, the ability to disrupt preformed biofilms of all four Candida species analyzed by the death of fungal cells wrapped by the extracellular matrix suggests a potential therapeutic role for PYED-1. In the eradication biofilm assay, exposure of preformed C. glabrata biofilms to 128, and 32 µg/mL of PYED-1 reduced the cell viability by 75-92% ( Figure 2A) and the biofi biomass by 30-70% ( Figure 2B), as compared to untreated controls. PYED-1 at 64 µg/m was able to reduce the number of viable C. tropicalis to over 80% (Figure 2A), while biofi biomass was diminished by 50% ( Figure 2B). In contrast, PYED-1 displayed a significa reduction in the number of viable cells of preformed biofilms of C. albicans and parapsilosis when compared to untreated controls, but only marginally reduced the biofi biomass ( Figure 2). Candida biofilms are widely considered to be a key determinant of t high mortality rate attributed to candidiasis [7]. Candida can form biofilms on biomedi implants that are difficult to eradicate by clinical drugs such as amphotericin fluconazole, flucytosine and itraconazole. They are also responsible for the high resistan to antifungal drugs [18]. Antifungal resistance due to biofilm formation is frequen reported in clinical settings, and it counteracts the achievement of effective treatme strategies [19]. Antibiofilm agents represent a novel approach to reducing antifung and biofilm biomass (B) were measured by XTT reduction assay and CV staining, respectively. All experiments were performed in triplicate. Asterisks indicate a statistically significant difference (** p-value ≤ 0.01 and *** p-value ≤ 0.001 from the nontreated controls without PYED-1).

PYED-1 Inhibited the Filamentation and Adhesion of C. albicans
Mature Candida biofilms are formed by a mixture of yeast cells, pseudohyphae and hyphae [21], and the dimorphic switch of yeast cells to hyphal cells is required for the maturation of structured biofilms by C. albicans [22]. The transition of yeast cells to hyphae is considered an important virulence factor of C. albicans: it can promote fungal invasion of human mucosal tissues, cause invasive fungal infections and aid in escape from immune cells [23]. Hyphal formation is associated with virulence, as genes encoding virulence factors are coregulated with hypha-specific genes [23]. The ability of PYED-1 to inhibit C. albicans filamentation at different concentrations after 5 h of incubation at 37 • C was investigated by microscopy ( Figure 3). maturation of structured biofilms by C. albicans [22]. The transition of yeast cells to hyphae is considered an important virulence factor of C. albicans: it can promote fungal invasion of human mucosal tissues, cause invasive fungal infections and aid in escape from immune cells [23]. Hyphal formation is associated with virulence, as genes encoding virulence factors are coregulated with hypha-specific genes [23]. The ability of PYED-1 to inhibit C. albicans filamentation at different concentrations after 5 h of incubation at 37 °C was investigated by microscopy ( Figure 3). The filamentation of C. albicans induced by serum was effectively suppressed in the presence of PYED-1 in RPMI-FBS (fetal bovine serum) medium in a concentrationdependent manner. After 5 h of drug exposure, PYED-1 fully inhibited hyphae formation of C. albicans ATCC 10,231 strain at 32 µg/mL, as compared to the untreated control. Microscopic images of C. albicans untreated or treated with 2 µg/mL and 4 µg/mL of PYED-1 formed long hyphae ( Figure 3A-C). In cells treated with 8 µg/mL and 16 µg/mL of the drug, fewer filaments and more yeasts per field were observed ( Figure 3D,E). Microscopic images of C. albicans cells treated with 32 µg/mL of PYED-1 showed yeast cells but no filaments ( Figure 3F). Many molecules induce changes in cell wall components associated with the transition from yeast to hyphal growth and/or reduce the expression of various virulence factors [24]. Molecules with antimicrobial activity, which exhibit a synergistic effect with conventional drugs or reduce the expression of pathogen virulence factors, can represent new treatment options that are effective in combating fungal infections [24,25]. The prevention of hyphae growth by PYED-1 is a significant result since The filamentation of C. albicans induced by serum was effectively suppressed in the presence of PYED-1 in RPMI-FBS (fetal bovine serum) medium in a concentrationdependent manner. After 5 h of drug exposure, PYED-1 fully inhibited hyphae formation of C. albicans ATCC 10231 strain at 32 µg/mL, as compared to the untreated control. Microscopic images of C. albicans untreated or treated with 2 µg/mL and 4 µg/mL of PYED-1 formed long hyphae ( Figure 3A-C). In cells treated with 8 µg/mL and 16 µg/mL of the drug, fewer filaments and more yeasts per field were observed ( Figure 3D,E). Microscopic images of C. albicans cells treated with 32 µg/mL of PYED-1 showed yeast cells but no filaments ( Figure 3F). Many molecules induce changes in cell wall components associated with the transition from yeast to hyphal growth and/or reduce the expression of various virulence factors [24]. Molecules with antimicrobial activity, which exhibit a synergistic effect with conventional drugs or reduce the expression of pathogen virulence factors, can represent new treatment options that are effective in combating fungal infections [24,25]. The prevention of hyphae growth by PYED-1 is a significant result since yeast-to-hypha transition is a critical virulence feature which mediates C. albicans invasion into infected tissue.
A hyperfilamentous strain of C. albicans is characterized by a high adhesion ability [26]. Hypha-specific genes encode proteins that are involved in the adhesion to host cells [23]. Strong adherence to cell/tissue or the surfaces of medical devices is the earliest stage of a Candida infection. Since PYED-1 influenced the yeast-to-hyphal morphological transition, we thought it interesting to check whether this compound could affect cell adhesion to abiotic surfaces. As shown in Figure 4, PYED-1 significantly reduced the adhesion of C. albicans ATCC 10,231 to polystyrene surface by up to 30% compared with the negative control. [23]. Strong adherence to cell/tissue or the surfaces of medical devices is the earliest stage of a Candida infection. Since PYED-1 influenced the yeast-to-hyphal morphological transition, we thought it interesting to check whether this compound could affect cell adhesion to abiotic surfaces. As shown in Figure 4, PYED-1 significantly reduced the adhesion of C. albicans ATCC 10,231 to polystyrene surface by up to 30% compared with the negative control. PYED-1 exhibited a 46% and 55% reduction in the adhesion of C. albicans at sub-MIC concentrations of 2 µg/mL and 4 µg/mL, respectively. At the highest concentration used (16 µg/mL), PYED-1 decreased about 70% of the adhesion, as compared to the untreated controls. PYED-1 can inhibit the adherence of C. albicans and the formation of hyphae. This suggests that PYED-1 may prevent the colonization and tissue penetration of C. albicans. Several studies have shown that adherence and hyphae formation are closely connected. The ALS gene family, encoding a set of cell glycoproteins that promote fungal adherence and biofilm formation, is expressed specifically during hyphal development [27]. Hwp1, a C. albicans cell-surface protein expressed only on hyphae, plays an important role in hyphal development, but is also required for fungal adherence and biofilm formation [27,28].
Blastospores, pseudohyphae and hyphae play a critical role in the process of C. albicans biofilm development and maturation, providing stability to the biofilm structure [23]. Therefore, inhibition of the adhesion of yeast cells and their differentiation in filamentous form can represent an effective therapeutic alternative [29]. In light of this, the glucocorticoid PYED-1 with a broad antimicrobial activity has the potential to serve as a preventive tool. It functions by affecting the adhesion of yeast cells and their differentiation in filamentous form, and by inhibiting existing or forming C. albicans biofilms. However, the specific antibiofilm mechanism of PYED-1 has not yet been described. Future studies will be necessary to understand the mode of action of PYED-1 and to evaluate its therapeutic use during Candida spp. infections. PYED-1 exhibited a 46% and 55% reduction in the adhesion of C. albicans at sub-MIC concentrations of 2 µg/mL and 4 µg/mL, respectively. At the highest concentration used (16 µg/mL), PYED-1 decreased about 70% of the adhesion, as compared to the untreated controls. PYED-1 can inhibit the adherence of C. albicans and the formation of hyphae. This suggests that PYED-1 may prevent the colonization and tissue penetration of C. albicans. Several studies have shown that adherence and hyphae formation are closely connected. The ALS gene family, encoding a set of cell glycoproteins that promote fungal adherence and biofilm formation, is expressed specifically during hyphal development [27]. Hwp1, a C. albicans cell-surface protein expressed only on hyphae, plays an important role in hyphal development, but is also required for fungal adherence and biofilm formation [27,28].

Fungal Strain, Culture Condition and Compound Synthesis
Blastospores, pseudohyphae and hyphae play a critical role in the process of C. albicans biofilm development and maturation, providing stability to the biofilm structure [23]. Therefore, inhibition of the adhesion of yeast cells and their differentiation in filamentous form can represent an effective therapeutic alternative [29]. In light of this, the glucocorticoid PYED-1 with a broad antimicrobial activity has the potential to serve as a preventive tool. It functions by affecting the adhesion of yeast cells and their differentiation in filamentous form, and by inhibiting existing or forming C. albicans biofilms. However, the specific antibiofilm mechanism of PYED-1 has not yet been described. Future studies will be necessary to understand the mode of action of PYED-1 and to evaluate its therapeutic use during Candida spp. infections.

Fungal Strain, Culture Condition and Compound Synthesis
Candida strains evaluated in this study included the C. albicans ATCC 10231 reference strain and 6-8 clinical isolates of each Candida species belonging to a bacterial collection that was previously established. No ethical approval was required for this study as there was no access to patients' data. Strains were routinely grown on Sabouraud dextrose agar (SDA) and in RPMI-1640 broth buffered with MOPS (pH 7.0) containing glucose 2% (RPMI-1640 2% G) at 37 • C. The chemical synthesis and structural characterization of PYED-1 were accomplished as previously reported [13]. Stock solutions of the compounds at the concentration of 50 mg/mL were prepared in dimethyl sulfoxide (DMSO) and kept at −20 • C and diluted in RPMI-1640 2% G broth just prior to the assays.

Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Fungicide Concentration (MFC)
PYED-1 was evaluated for its in vitro antifungal activities according to EUCAST guidelines using the two-fold serial dilution technique in 96-well microtiter plates [30]. Briefly, yeasts from overnight cultures on SDA were suspended in RPMI-1640 2% G broth Antibiotics 2021, 10, 1396 7 of 9 to get 1-5 × 10 5 colony-forming units (cfu)/mL. Two-fold serial dilutions of PYED-1 were prepared in RPMI-1640 2% G medium, starting from 512 to 1 µg/mL, in order to obtain a 2× final concentration of the antifungal agent. Each well of plate was inoculated with 100 µL of 1-5 × 10 5 cfu/mL yeast suspension and 100 µL of twice the final antifungal agent PYED-1 or 100 µL of sterile drug-free medium (growth control). A sterility control containing 200 mL RPMI-1640 medium was included in each plate. The plate was incubated without agitation at 37 • C for 24-28 h. The optical density at 450 nm was measured using a microplate reader (Bio-Rad Laboratories S.r.l., Segrate (MI) -Italy). MIC was the lowest concentration inhibiting ≥90% growth when compared to growth control. The effect of different concentrations of DMSO (ranging from 0.1% to 0.5%) on fungal growth was separately tested. In order to evaluate the minimum lethal concentration (MLC, i.e., the lowest concentration showing a growth inhibition of 100%), 20 µL of yeast suspensions from wells without visible growth were transferred to SDA plates. These plates were incubated at 37 • C and checked for growth after 24 h. All experiments were performed in triplicate and were repeated independently at least three times.

Checkboard Microdilution Assay
The checkerboard method was used to evaluate the MICs for PYED-1 alone and in combination with the antifungal agents fluconazole and posaconazole for C. parapsilosis 84,609 and 84,614, C. glabrata 61,115 and C. albicans 61,446 and 61,691 strains, as described previously [31]. The plates were incubated at 37 • C for 24-28 h. The fractional inhibitory concentration index was calculated for the compound combinations, with a FICI < 0.5 indicating "synergism", a FICI > 4 indicating "antagonism", and a FICI between 0.5 and 4 defined as "without interaction" [32]. The results were obtained from tests that were repeated three times.

Germ Tube Inhibition Assay
To determine serum-induced cell filamentation, C. albicans ATCC 10,231 cells were inoculated in 2 mL of RPMI-1640 medium containing 10% (v/v) FBS at a density of 10 6 cfu/mL in a glass tube without (control) or with the addition of PYED-1 solutions at 2, 4, 8, 16 and 32 µg/mL. After a 5 h incubation at 37 • C, 30 µL cells from treated and untreated samples were smeared, fixed, stained with 0.3% crystal violet and observed under a light microscope.

Adhesion Assay
The adhesion prevention efficacy of PYED-1 was assayed using the crystal violet biofilm staining method [33]. Cultures of C. albicans ATCC 10,231 were grown in RPMI-1640 2% G and incubated overnight at 30 • C with agitation (100 rpm). Cells were harvested and washed with PBS and diluted to a density of 1 × 10 6 cfu/mL in RPMI-1640 2% G. The wells of polystyrene flat-bottom 96-well microtiter plates were conditioned with 50% FBS in PBS for at least 30 min at room temperature. Then, the FBS was aspirated and wells were rinsed once with PBS. One hundred microliters of the yeast suspension was added to each well containing 100 µL of twice the final concentration of antifungal agent PYED-1 (2, 4, 8, 16 and 32 µg/mL) or 100 µL of sterile drug-free medium (growth control). The plate was incubated at 37 • C for 90 min. Then, the medium was aspirated, wells were washed with PBS twice and 100 µL of 0.1% crystal violet was added to each well. After 15 min, the plate was washed two times and CV was dissolved with 200 µL of 100% ethanol. Absorbance was measured at 595 nm using a microplate reader (Bio-Rad Laboratories S.r.l.). The percentage of biofilm mass reduction was calculated as [(Ac−At)/Ac] × 100, where Ac is the OD 595 for the control well and At is the OD 595 for the biofilm in the presence of PYED-1. All data points are expressed as means + SDs of three separate experiments performed in triplicate.

Anti-Preformed-Biofilm Assay
Biofilms were allowed to form in each well as described in the adhesion assay and were treated following 24 h of incubation. After the adhesion step, nonadherent cells were removed and wells were washed with PBS. Two hundred microliters of RPMI-1640 2% G was added to each well and subsequently incubated at 37 • C for 18-24 h in an orbital shaker (100 rpm). After incubation, wells were washed with PBS and 200 µL of twice the final antifungal agent PYED-1 (64, 128 and 256 µg/mL) or 100 µL of sterile drug-free medium (growth control) was added. Biofilm was incubated statically at 37 • C for an additional 18-24 h. After PYED-1 treatment, the metabolic activities of biofilm cells and biofilm biomass were measured by XTT reduction assay and CV staining [34], respectively. Briefly, 200 µL of a solution of XTT (0.5 mg/mL in PBS; Sigma, Milan, Italy) menadione (1 µM in acetone; Sigma) was added to each prewashed well. Following incubation in the dark for up to 2 h at 37 • C, the biofilm metabolic activity was determined through the measurement of the absorbance value at 490 nm using a microplate reader (Bio-Rad Laboratories S.r.l.). Viability values were compared with respect to control samples treated with scalar doses of DMSO (concentrations ranging from 0.064% to 0.256%). All data points are expressed as means + SDs of three separate experiments performed in triplicate.

Statistical Analysis
All statistical analyses were performed with GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). All results are presented as arithmetic means ± standard deviations. Statistical differences between PYED-1 treated and untreated samples were analyzed by one-way analysis of variance (ANOVA) followed by Student's t-test. Statistical significance was considered at p-value of <0.05.