Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.


Introduction
Candida albicans is an opportunistic fungal pathogen responsible for high morbidity and mortality in immunocompromised patients [1]. The most common class of antifungal drugs used for the treatment of candidiasis are azoles, which inhibit ergosterol biosynthesis by targeting cytochrome P-450 lanosterol 14α-demethylase (CYP51A1 and Erg11p) [2]. However, infections caused by C. albicans are recurrent and difficult to treat due to the ability of fungal cells to acquire a multidrug-resistant (MDR) phenotype [3]. In 2019, it was estimated that up to 20% of clinical Candida spp. isolates exhibit azole resistance [4].
One of the underlying processes responsible for MDR in C. albicans is the increased synthesis of membrane efflux transporters such as Cdr1p and Cdr2p, which belong to the ATP-Binding Cassette (ABC) family, and Mdr1p, which belongs to the Major Facilitator Superfamily (MFS) [5]. Although each transporter possesses different substrate specificities, all three can export fluconazole (FLC), the most common therapeutic anticandidal azole [5].
Candidiasis is associated with several diseases, including diabetes [6]. Nearly 50% of all Candida spp. have been identified in oral cavity samples from prediabetic patients [7]. It has been estimated that 80-90% of people with type I diabetes are carriers of Candida spp., and 70% of them are likely to develop infections of the skin and mucous membranes [8]. Type II diabetics are less likely to experience oral and mucosal Candida colonization but 10-fold more susceptible to inner organ infections [8,9]. The risk of C. albicans infection is increased in diabetic patients, partially due to increased serum glucose levels [8]. Glucose has already been described to promote the growth of C. albicans, as well as the Hog1-mediated resistance to oxidative and cationic stresses and increased resistance towards antifungal drugs [10][11][12]. We previously reported that glucose induces CDR1 gene expression in a C.

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albicans parental strain and induces the de novo synthesis of Cdr1p in a C. albicans cdr2∆ mutant [13].
Recent studies have reported an increase in fructose concentration in the serum of diabetes patients [14,15], as well as accompanying several oncological diseases [16]. Fructose overabsorption is a known etiological factor of diabetes mellitus type II or metabolic syndrome [17] and is reported to promote germ tube formation and the adherence of C. albicans to epithelial mucous surfaces [18]. However, to date, no investigations concerning the influence of fructose on either the antifungal resistance or MDR transporter activity in C. albicans have been reported.
In this study, we postulate that fructose promotes a FLC resistance in C. albicans due to the activation of Cdr1p and by inducing the de novo synthesis of Mdr1p. We propose that elevated serum fructose levels may be one of the factors responsible for Mdr1p production in C. albicans cells.

Fructose as a Carbon Source Promotes FLC Resistance and Increased Efflux Activities of Cdr1p and Mdr1p
As a Crabtree microorganism, C. albicans assimilates different carbon sources at the same time, which is an adaptation to host niches where the nutrient availability may vary [19][20][21]. Fluctuations in the availability of various carbon sources have a profound effect on the physiology of C. albicans, including changes in the gene expression, which may result in drug resistance [22]. Here, we aimed to evaluate the effect of fructose, a carbon source naturally occurring in the human oral cavity, intestines, and blood plasma [15][16][17]19], on the sensitivity of C. albicans to FLC.
Firstly, the growth of the C. albicans CAF2-1 strain in the presence of different concentrations of FLC in the YNB-based media containing glucose or fructose as a sole carbon source ( Figure 1A). The growth of glucose-grown cells was inhibited ≥50% in the presence of 1-µg/mL FLC. Fructose-grown cells exhibited higher FLC tolerance, and ≥50% growth inhibition was observed only at a 4-µg/mL FLC concentration. At 8-µg/mL FLC and above, the growth of the glucose-grown cells was inhibited 80%, whereas the growth of the fructose-grown cells was inhibited only 60% ( Figure 1A).
The activity of the MDR transporters is a primary factor contributing to the increased FLC tolerance among C. albicans isolates [23]. In order to determine the role of MDR transporters in the fructose-induced FLC resistance, we analyzed the growth phenotype of a set of isogenic C. albicans strains lacking one or more of the MDR transporters ( Figure 1B). Clinical C. albicans strains isolated from patients treated with FLC display a high expression of all three transporters [24]; however, using standard laboratory conditions (general media and C. albicans reference strains), no gene expression or production of Cdr2p or Mdr1p are detectable [24].
In the YNB media containing glucose, the growth of parental C. albicans CAF2-1 cells was partially inhibited at 1-µg/mL FLC. Increasing the FLC concentration to 2 µg/mL further intensified this inhibition. A similar growth phenotype to that of CAF2-1 was observed in the DSY653 (cdr2∆) and DSY465 (mdr1∆) strains. However, only the residual growth of DSY448 (cdr1∆), DSY654 (cdr1∆cdr2∆), and DSY1050 (cdr1∆cdr2∆mdr1∆) was observed, regardless of the FLC concentration used. This suggests that, in glucose-grown C. albicans cells, Cdr1p is primarily responsible for the FLC tolerance [25,26]. This is in agreement with previously published data, where deletion of the CDR1 gene vastly sensitized C. albicans towards FLC [26,27]. Under the same conditions, the deletion of CDR2 or MDR1 did not influence the FLC tolerance in C. albicans using glucose-based media [26,27]. The reason was most likely related to the fact that the gene promoters of CDR2 and MDR1 are lacking a basal expression element (BEE), which is only present within the gene promoter of CDR1 [28,29], while the synthesis of Cdr2p and Mdr1p is only induced by external factors such as azoles, fluphenazine, and β-estradiol in the case of Cdr2p or benomyl and H 2 O 2 in the case of Mdr1p [28][29][30]. It has been reported that . Cells were cultured with either yeast nitrogen base glucose (YNBG) or yeast nitrogen base fructose (YNBF) media for 24 h at 28 °C (mean ± SD, n = 3). Statistical analysis was performed by comparing the percentage growth between YNBG-and YNBF-grown cells at the same FLC concentrations (*, p < 0.05 and **, p < 0.01). (B) Growth phenotypes of C. albicans CAF2-1 (parental strain), DSY448 (cdr1Δ), DSY653 (cdr2Δ), DSY465 (mdr1Δ), DSY654 (cdr1Δcdr2Δ), and DSY1050 (cdr1Δcdr2Δmdr1Δ) strains after 48-h incubation at 28 °C. All strains were grown on either YNBG or YNBF media in the presence of a range of FLC concentrations (0-2 µg/mL).
The activity of the MDR transporters is a primary factor contributing to the increased FLC tolerance among C. albicans isolates [23]. In order to determine the role of MDR transporters in the fructose-induced FLC resistance, we analyzed the growth phenotype of a set of isogenic C. albicans strains lacking one or more of the MDR transporters ( Figure 1B). Clinical C. albicans strains isolated from patients treated with FLC display a high expression of all three transporters [24]; however, using standard laboratory conditions (general media and C. albicans reference strains), no gene expression or production of Cdr2p or Mdr1p are detectable [24].
To confirm these conclusions, we evaluated the efflux activities of MDR pumps using the same set of C. albicans strains grown in either glucose-or fructose-containing media ( Figure 2). For this purpose, we used two fluorescent dyes: rhodamine 6G (R6G), which is a substrate of Cdr1p and Cdr2p but not Mdr1p, and Nile red (NR), which is a substrate of Cdr1p and Mdr1p but not Cdr2p [32]. Both the fluorescent substrates accumulate within the yeast cells and are actively removed by the transporters, which is measured as an extracellular fluorescence, and reflects the efflux activity of the transporters [32]. The efflux of R6G was observed only in the case of C. albicans strains that contain Cdr1p (CAF2-1, DSY653, and DSY465), regardless of the carbon source used ( Figure 2A). However, in these strains, the R6G efflux was ~3.5-fold higher in the media containing fructose (Figure 2A). Based on these observations, we concluded that Cdr2p is probably not activated on either glucose-or fructose-containing media, and Cdr1p activity is higher in the case of fructose-grown cells.
The efflux of NR in glucose-grown cells was observed in strains expressing Cdr1p (CAF2-1, DSY653, and DSY465) a comparable level ( Figure 2B) to the efflux of R6G at the same carbon source (Figure 2A). The NR efflux in fructose-grown cells was observed in all C. albicans strains except for DSY1050, which is deficient in all MDR transporters. The C. albicans strains that contain both Cdr1p and Mdr1p (CAF2-1 and DSY653) were char- The efflux of R6G was observed only in the case of C. albicans strains that contain Cdr1p (CAF2-1, DSY653, and DSY465), regardless of the carbon source used ( Figure 2A). However, in these strains, the R6G efflux was~3.5-fold higher in the media containing fructose (Figure 2A). Based on these observations, we concluded that Cdr2p is probably not activated on either glucose-or fructose-containing media, and Cdr1p activity is higher in the case of fructose-grown cells.
The efflux of NR in glucose-grown cells was observed in strains expressing Cdr1p (CAF2-1, DSY653, and DSY465) a comparable level ( Figure 2B) to the efflux of R6G at the same carbon source (Figure 2A). The NR efflux in fructose-grown cells was observed in all C. albicans strains except for DSY1050, which is deficient in all MDR transporters. The C. albicans strains that contain both Cdr1p and Mdr1p (CAF2-1 and DSY653) were characterized by a~6-fold higher NR efflux when grown on the fructose-containing medium. The strains that contain Mdr1p but not Cdr1p (DSY448 and DSY654) displayed a~5-fold higher NR efflux when grown in the fructose-containing medium. The strain lacking Mdr1p but expressing Cdr1p (DSY465) was characterized by a~2.5-fold higher NR efflux when grown on fructose. This suggested that fructose-grown cells, despite a higher Cdr1p-dependent efflux activity, additionally feature an active Mdr1 transporter.

Fructose-Grown Cells Are Characterized by High Levels of Cdr1p and Mdr1p
The results described in Section 2.1, together with previous reports, suggest that, in the case of glucose-grown C. albicans cells, Cdr1p is primarily responsible for the FLC tolerance, with negligible roles played by Cdr2p or Mdr1p [26,27]. However, in C. albicans cells grown on fructose, particularly in the case of strains positive for the MDR1 gene but negative for the CDR1 gene, we observed a high FLC tolerance ( Figure 1B) and high NR efflux ( Figure 2B). We concluded that these observations may result from the synthesis of Mdr1p in C. albicans cells as a result of the growth in the presence of fructose. In order to confirm those conclusions, we constructed a series of GFP-tagged C. albicans strains. We labeled Cdr1p-GFP in the cdr2∆ or mdr1∆ backgrounds; Cdr2p-GFP in the cdr1∆ or mdr1∆ backgrounds; and Mdr1p-GFP in the cdr1∆, cdr2∆, or cdr1∆cdr2∆ backgrounds (Table 1). We performed microscopic observations of the fluorescent signal in the constructed C. albicans strains grown in either glucose or fructose media, which were further validated by Western blotting (Figures 3 and 4).
The results described in Section 2.1, together with previous reports, suggest t the case of glucose-grown C. albicans cells, Cdr1p is primarily responsible for th tolerance, with negligible roles played by Cdr2p or Mdr1p [26,27]. However, in C. a cells grown on fructose, particularly in the case of strains positive for the MDR1 ge negative for the CDR1 gene, we observed a high FLC tolerance ( Figure 1B) and hi efflux ( Figure 2B). We concluded that these observations may result from the synth Mdr1p in C. albicans cells as a result of the growth in the presence of fructose. In o confirm those conclusions, we constructed a series of GFP-tagged C. albicans strai labeled Cdr1p-GFP in the cdr2Δ or mdr1Δ backgrounds; Cdr2p-GFP in the cd mdr1Δ backgrounds; and Mdr1p-GFP in the cdr1Δ, cdr2Δ, or cdr1Δcdr2Δ backgr (Table 1). We performed microscopic observations of the fluorescent signal in th structed C. albicans strains grown in either glucose or fructose media, which were f validated by Western blotting (Figures 3 and Figure 4). The Cdr1p-GFP signal was observed in the plasma membranes of C. albican the aforementioned conditions ( Figure 3A). However, the Cdr1p-GFP signal was stronger in fructose-grown cells than in glucose-grown cells. A Western blotting p analysis ( Figure 3C) confirmed those observations ( Figure 3C). Additionally, it wa cluded that the absence of detectable Cdr2p or Mdr1p does not influence the le Cdr1p ( Figure 3A). In contrast, we previously observed that the presence of gluc creased the Cdr1p levels in the C. albicans cdr2Δ strain to a greater extent than in t transporter homologous of Cdr1p in Saccharomyces cerevisiae, a compensatory activat of other ABC transporters (Snq2 and Yor1) occurs. Similar observations were reported ABC transporters of pathogenic fungus Trichophyton spp. [34]. In C. albicans, the tr scriptional regulation of the CDR1 and CDR2 genes overlaps: Mrr2, Upc2, Ndt80, Znc1 act as positive regulators [35,36], and Flo8 as a negative regulator [36] of both gen However, our results suggest that the disruption of either C. albicans ABC transpor does not induce the production of the remaining protein ( Figure 3). Cells cultured in a medium with glucose, in contrast to those cultured with fruct did not synthesize Mdr1p, which was the reason for the absence of the Mdr1p-GFP sig during the microscopic observations and the lack of Mdr1p detected by Western ( Figure 4). Moreover, in the C. albicans KS073 strain, which lacks both the Cdr1 and C proteins, we observed a more pronounced Mdr1p-GFP signal. Based on these obser tions, we concluded that C. albicans cells synthesize Mdr1p in the presence of fruct without any other stimulating factor ( Figure 4B).
The promoter of the MDR1 gene includes a H2O2 responsive element (HRE), wh induces the production of Mdr1p upon oxidative stress [37], which led us to hypothe that the fructose metabolism might induce oxidative stress. Conversely, however, fr tose has been described to exhibit a general protective effect against oxidative stress i cerevisiae cells [38], including a specific protection against H2O2 and reactive oxygen s cies (ROS) [39]. This led us to a different hypothesis. In eukaryotic cells, fructose, glucose, is metabolized to pyruvate, which supplies energy to cells through the Kr cycle [40]. However, fructose has been described to also be metabolized to a toxic gly lytic byproduct called methylglyoxal (MG), the elevated levels of which are respons for hepatotoxicity in diabetic patients [40]. In Candida lusitaniae, the MG metabolism believed to be mediated by Mgd1 and Mgd2 reductases, the expression of which is c trolled by Mrr1p, which, in turn, is inducible by MG [41]. The homologous protein in albicans (CaMrr1p) is a major transcriptional inductor of MDR1 [42]. Thus, we hypo size that the fructose metabolism, leading to the production of MG, might directly ind expression of the MDR1 gene and production of Mdr1p through the activation of Mrr The Cdr1p-GFP signal was observed in the plasma membranes of C. albicans in all the aforementioned conditions ( Figure 3A). However, the Cdr1p-GFP signal was visibly stronger in fructose-grown cells than in glucose-grown cells. A Western blotting protein analysis ( Figure 3C) confirmed those observations ( Figure 3C). Additionally, it was concluded that the absence of detectable Cdr2p or Mdr1p does not influence the level of Cdr1p ( Figure 3A). In contrast, we previously observed that the presence of glucose increased the Cdr1p levels in the C. albicans cdr2∆ strain to a greater extent than in the parental strain [13]. It must be noted that, previously, we performed only short-term glucose induction (12 or 36 min). It may therefore be concluded that short-time exposure of the C. albicans cdr2∆ strain to glucose results an increased production of Cdr1p, while, after long-term incubation with glucose, the Cdr1p protein level eventually stabilizes.
The Cdr2p-GFP signal was not detected under either of the experimental conditions ( Figure 3B,D). This shows that, regardless of the carbon source (glucose or fructose), Cdr2p was not involved in either the FLC resistance ( Figure 1) or R6G efflux ( Figure 2A). Additionally, this explains the lack of differences in R6G efflux between the parental CAF2-1 and DSY653 (cdr2∆) strains ( Figure 2A). Additionally, we observed that the absence of Cdr1p or Mdr1p does not induce the production of Cdr2p. Kolaczkowska et al. [33] previously reported that, upon disruption of the PDR5 gene, which encodes an ABC transporter homologous of Cdr1p in Saccharomyces cerevisiae, a compensatory activation of other ABC transporters (Snq2 and Yor1) occurs. Similar observations were reported for ABC transporters of pathogenic fungus Trichophyton spp. [34]. In C. albicans, the transcriptional regulation of the CDR1 and CDR2 genes overlaps: Mrr2, Upc2, Ndt80, and Znc1 act as positive regulators [35,36], and Flo8 as a negative regulator [36] of both genes. However, our results suggest that the disruption of either C. albicans ABC transporters does not induce the production of the remaining protein ( Figure 3).
Cells cultured in a medium with glucose, in contrast to those cultured with fructose, did not synthesize Mdr1p, which was the reason for the absence of the Mdr1p-GFP signal during the microscopic observations and the lack of Mdr1p detected by Western blot (Figure 4). Moreover, in the C. albicans KS073 strain, which lacks both the Cdr1 and Cdr2 proteins, we observed a more pronounced Mdr1p-GFP signal. Based on these observations, we concluded that C. albicans cells synthesize Mdr1p in the presence of fructose without any other stimulating factor ( Figure 4B).
The promoter of the MDR1 gene includes a H 2 O 2 responsive element (HRE), which induces the production of Mdr1p upon oxidative stress [37], which led us to hypothesize that the fructose metabolism might induce oxidative stress. Conversely, however, fructose has been described to exhibit a general protective effect against oxidative stress in S. cerevisiae cells [38], including a specific protection against H 2 O 2 and reactive oxygen species (ROS) [39]. This led us to a different hypothesis. In eukaryotic cells, fructose, like glucose, is metabolized to pyruvate, which supplies energy to cells through the Krebs cycle [40]. However, fructose has been described to also be metabolized to a toxic glycolytic byproduct called methylglyoxal (MG), the elevated levels of which are responsible for hepatotoxicity in diabetic patients [40]. In Candida lusitaniae, the MG metabolism is believed to be mediated by Mgd1 and Mgd2 reductases, the expression of which is controlled by Mrr1p, which, in turn, is inducible by MG [41]. The homologous protein in C. albicans (CaMrr1p) is a major transcriptional inductor of MDR1 [42]. Thus, we hypothesize that the fructose metabolism, leading to the production of MG, might directly induce expression of the MDR1 gene and production of Mdr1p through the activation of Mrr1p.

Serum Levels of Fructose Induces de Novo Synthesis of Mdr1p and Enhanced Synthesis of Cdr1p
The concentrations of glucose and fructose in different niches of the human bodyspecifically, the digestive tract and bloodstream-depend mostly on dietary factors. The ingestion of sugar-rich products may lead to an increase in fructose concentration in the peripheral venous blood of up to~0.006%, and the glucose concentration of up tõ 0.2% [19,43]. Rodaki et al. [10] reported that a short-term exposure of C. albicans to 0.1% glucose induces a stress response, which includes the transcriptional activation of CDR1.
We report that C. albicans cells, grown up until the early logarithmic phase with fructose as the sole carbon source, are characterized by the presence of Mdr1p (Figure 4) and increased levels of Cdr1p ( Figure 3). We aimed to investigate whether this effect occurs upon the short-term exposure of C. albicans to glucose, fructose, or both sugars in the bloodstream. To this end, we cultured the C. albicans KS052 (Cdr1p-GFP) and KS070 (Mdr1p-GFP) strains in YNBG medium until the early logarithmic phase; at which point, we induced cell starvation by incubating cells for one hour in a HEPES-NaOH buffer. The starved cells were supplemented with either glucose (0.2%), fructose (0.006%), or both sugars and analyzed for the expression of the Cdr1 and Mdr1 proteins ( Figure 5).
We observed that C. albicans KS052 cells exposed to glucose or a glucose-fructose mixture are characterized by pronounced Cdr1p-GFP fluorescence in the plasma membrane ( Figure 5A). Western blotting revealed an increasing Cdr1p-GFP signal proportionate to the increase in incubation time with glucose or a glucose-fructose mixture ( Figure 5C). This suggests an increase in Cdr1p synthesis induced by glucose, which is in agreement with the data reported by Rodaki et al. [10] and Szczepaniak et al. [13]. However, we observed a slightly more pronounced fluorescence of Cdr1p-GFP in cells exposed to fructose, as well as a slightly increased signal seen with Western blotting ( Figure 5).
We observed no Mdr1p-GFP signal at the beginning of the induction (time = 0 min), which is in agreement with the data presented in Figure 4. The exposure of the C. albicans KS070 strain to glucose did not lead to the synthesis of Mdr1p-GFP. Only exposure to fructose induced a detectable Mdr1p-GFP signal ( Figure 5). Thus, it may be concluded that the exposure of C. albicans to low concentrations of fructose induces a de novo synthesis of Mdr1p after only 30 min of exposure.
We conclude that fructose as a carbon source induces FLC resistance in C. albicans in laboratory conditions in a general culture media. However, our observations may be of particular importance, as enhanced concentrations of fructose in the bloodstream persist for a much longer time than glucose (~three hours after fructose ingestion) before returning to the baseline levels [43]. brane ( Figure 5A). Western blotting revealed an increasing Cdr1p-GFP signal p tionate to the increase in incubation time with glucose or a glucose-fructose m ( Figure 5C). This suggests an increase in Cdr1p synthesis induced by glucose, whic agreement with the data reported by Rodaki et al. [10] and Szczepaniak et al However, we observed a slightly more pronounced fluorescence of Cdr1p-GFP i exposed to fructose, as well as a slightly increased signal seen with Western bl ( Figure 5).

Conclusions
These findings demonstrate that fructose as a carbon source enhances the FLC resistance in Candida albicans by two modes: the activation of Mdr1p and by inducing elevated levels of Cdr1p. We observed that fructose-grown C. albicans cells have a higher efflux activity of both transporters as opposed to glucose-grown cells, which constitutively synthesize only Cdr1p. Additionally, we concluded that the fructose serum level of 0.006% induces the de novo production of Mdr1p.

Strains and Growth Conditions
The C. albicans strains used in this study are listed in Table 1. CAF2-1, DSY448, DSY653, DSY465, DSY654, and DSY1050 were kind gifts from Professor D. Sanglard (Lausanne, Switzerland). KS052 and KS068 were previously constructed by our group, while KS053, KS054, KS064, KS065, KS070, KS073, KS074, and KS075 were constructed for the purposes of this study. Strains were pregrown at 28 • C on yeast nitrogen base glucose (YNBG) or yeast nitrogen base fructose (YNBF) media (0.67% YNB containing 2% glucose or 2% fructose, respectively) in an incubator while shaking at 120 rpm. Agar was added at a final concentration of 2% for medium solidification.
For most of the experiments, cells were grown until they reached the early logarithmic phase (8 h). Growth phases were determined as previously described [44]. Cells were centrifuged at 4500 rcf (relative centrifugal force) for 5 min; washed twice (4500 rcf, 5 min) with either phosphate-buffered saline (PBS), H 2 O dd , or 50-mM HEPES-NaOH buffer (pH 7.0); and resuspended in either PBS, H 2 O dd , or HEPES-NaOH to the indicated A 600 . For the induction experiments, C. albicans suspensions in HEPES-NaOH (A 600 = 1.0 in 25 mL) were incubated for 60 min at 28 • C. Subsequently, the cells were centrifuged at 4500 rcf for 5 min, washed twice (4500 rcf, 5 min) with HEPES-NaOH, and resuspended in HEPES-NaOH. Lastly, the cells were treated with glucose (0.2%), fructose (0.006%), or both sugars for either 30 or 60 min.

Percentage of Growth
To assess the effects of FLC on C. albicans growth in the presence of different carbon sources, we followed the protocol described by the Clinical and Laboratory Standards Institute (2008), 3rd ed. M27-A3 [49] with modifications. Briefly, 10-mg/mL stock solution of FLC was serially diluted in YNBG or YNBF media using 96-well sterile plates (Sarstedt, Nümbrecht, Germany). The various media compositions were then inoculated with C. albicans suspensions (final A 600 = 0.01 per well) and prepared in fresh YNBG or YNBF media from 24-h YNBG cultures. After 24 h of incubation at 28 • C, A 600 was measured using a ASYS UVM 340 microplate reader (Biogenet, Józefów, Poland). The percentage of growth of the C. albicans CAF2-1, DSY448, DSY653, DSY465, DSY654, and DSY1050 strains was determined by normalizing A 600 to that observed under conditions without FLC.

Efflux Activity of MDR Transporters
The efflux assay was performed according to the protocol of Szczepaniak et al. [50] with modifications. Briefly, 25-mL C. albicans suspensions (A 600 = 1.0 in 25-mL HEPES-NaOH) were treated with 5-mM 2-deoxy-D-glucose and incubated at 28 • C for 60 min with shaking at 200 rpm. Subsequently, 10-µM R6G or 7-µM NR were added before further incubation at 28 • C for 90 min with shaking at 200 rpm. Following this, the cells were centrifuged at 4500 rcf for 5 min, washed twice (4500 rcf, 5 min) with HEPES-NaOH, concentrated to 2 mL in HEPES-NaOH (A 600 = 10), and incubated at 28 • C for 5 min with shaking at 200 rpm. For each condition, the dye uptake was always ≥95%. Intensities of fluorescence (FIs) were measured 30 min after efflux. The assay was performed using a Cary Eclipse spectrofluorometer (Agilent Technologies, Santa Clara, CA, USA). The probes were excited at 529 nm (Ex slit = 5 nm), and emission was recorded at 553 nm (Em slit = 10 nm). IFs were normalized to 1 for the efflux activity of the control conditions (parental strain grown in YNBG).

Microscopic Studies
The CDR1-GFP, CDR2-GFP, or MDR1-GFP strains were suspended in PBS, concentrated, and observed under a Zeiss Axio Imager A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp (Zeiss, Poznań, Poland).

Western Blotting
Crude protein extracts from CDR1-GFP, CDR2-GFP, or MDR1-GFP strains were isolated as previously described [44,50]. Electrophoretic separation and transfer of Cdr1p-GFP was performed as previously described [50]. For Mdr1p-GFP separation, the following modification was applied: crude proteins from MDR1-GFP strains were separated on 8% SDS-polyacrylamide gels. For detection, mouse αGFP primary antibodies were used, followed by HRP-conjugated rabbit anti-mouse secondary antibodies. The remaining steps were performed as described in Reference [50].

Statistical Analysis
Unless stated otherwise, data represent the means ± standard errors from at least 3 biological replicates. Microscopic observations and Western blot analyses were performed at least in 2 independent replicates, of which the representatives were included in the figures. Statistical significance was determined using a Student's t-test (binomial, unpaired).
Author Contributions: Conceptualization, J.S. and A.K.; methodology, investigation, funding, and writing-original draft preparation, J.S.; and writing-review and editing and supervision, A.K. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.