The Transcription Factor Sfp1 Regulates the Oxidative Stress Response in Candida albicans

Candida albicans is a commensal that inhabits the skin and mucous membranes of humans. Because of the increasing immunocompromised population and the limited classes of antifungal drugs available, C. albicans has emerged as an important opportunistic pathogen with high mortality rates. During infection and therapy, C. albicans frequently encounters immune cells and antifungal drugs, many of which exert their antimicrobial activity by inducing the production of reactive oxygen species (ROS). Therefore, antioxidative capacity is important for the survival and pathogenesis of C. albicans. In this study, we characterized the roles of the zinc finger transcription factor Sfp1 in the oxidative stress response against C. albicans. A sfp1-deleted mutant was more resistant to oxidants and macrophage killing than wild-type C. albicans and processed an active oxidative stress response with the phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1 and high CAP1 expression. Moreover, the sfp1-deleted mutant exhibited high expression levels of antioxidant genes in response to oxidative stress, resulting in a higher total antioxidant capacity, glutathione content, and glutathione peroxidase and superoxide dismutase enzyme activity than the wild-type C. albicans. Finally, the sfp1-deleted mutant was resistant to macrophage killing and ROS-generating antifungal drugs. Together, our findings provide a new understanding of the complex regulatory machinery in the C. albicans oxidative stress response.


Introduction
Candida albicans (C. albicans) is a member of the human microbiota that normally inhabits the skin and mucosal surfaces of healthy individuals [1]. However, C. albicans is also an opportunistic pathogen that causes a wide range of infections including life-threatening hematogenously disseminated candidiasis, particularly in immunocompromised patients [1]. In addition to infections, C. albicans drug resistance has also emerged as a serious problem in clinical settings [2].
During the infection process and clinical therapy, the ability of C. albicans to adapt and respond to oxidative stress is critical for cell survival and virulence [3,4]. For example, C. albicans copes with reactive oxygen species (ROS) generation during the respiratory burst in phagocytic cells such as macrophages and neutrophils [3,5]. In addition, antifungal agents including amphotericin B, miconazole, and caspofungin induce ROS formation against C. albicans [6][7][8]. Previous studies showed that miconazole-mediated fungicidal activity against C. albicans was significantly inhibited by the addition of antioxidant [7], and superoxide dismutase inhibitors enhanced the activity of miconazole against C. albicans biofilm cells [9,10].

Reverse Transcription (RT) Real-Time Quantitative PCR (qPCR)
Total RNA extraction, cDNA synthesis by reverse transcription and real-time qPCR were performed as previously described [23]. The primers used in this study are listed in Table S2. The PMA1 transcript was used as an internal control for the RNA input [24]. The relative fold change in the expression of each gene was calculated using the 2 −∆∆CT method [25].

Extraction and Quantification of Total Protein
Cells were grown in SC medium with or without hydrogen peroxide (H 2 O 2 ) or menadione for 2 h, washed with phosphate buffered saline (PBS), and mixed with ice-cold protein extraction buffer as indicated for each assay described below. The cell suspensions were further mixed with 0.3 g acid-washed glass beads, disrupted by vortexing for 30 s, and immediately placed on ice for 30 s. This process was repeated eight times. Soluble proteins were collected by centrifugation (13,000× g) at 4 • C and quantified using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) based on the Bradford method.

Determination of Total Antioxidant Capacity
Total antioxidant capacity was measured using the Antioxidant Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. This assay relies on the ability of cellular antioxidants to inhibit the oxidation of ABTS (2,2 -azino-di-[3-ethylbenzthiazoline sulfonate]). Protein extraction was performed as described above using an extraction buffer containing 5 mM potassium phosphate (pH 7.4), 0.9% sodium chloride, and 0.1% glucose. Briefly, 10 µL of proteins was mixed with 150 µL ABTS, followed by the addition of 40 µL H 2 O 2 and 10 µL metmyoglobin to initiate the reaction. After incubation at room temperature for 5 min, the absorbance at 750 nm was measured spectrophotometrically. The total antioxidant capacity to prevent ABTS oxidation was compared with that of Trolox, a water-soluble tocopherol analogue. The total antioxidant capacity was expressed as molar Trolox equivalents.

Cell Susceptibility to Oxidants and Measurement of Intracellular ROS
Cell susceptibility to oxidants was examined by spot assay and propidium iodide (PI) staining as previously described [26]. In the spot assay, one colony was inoculated into YPD medium and grown at 30 • C overnight. Cells were collected by centrifugation, washed, and resuspended in sterile ddH 2 O. Cells were 10-fold serially diluted (3 × 10 7 to 3 × 10 3 cells/mL) and 5 µL of each sample was spotted onto SC or YPD agar plates containing H 2 O 2 or menadione. The plates were incubated at 30 • C for 4-5 days and photographed every day. For PI staining, cells were treated with H 2 O 2 or menadione for 2 h, harvested by centrifugation, washed with PBS, and resuspended in a PI staining solution (4 µg/mL PI in PBS). PI-positive cells were measured using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Intracellular ROS were detected using cell permeable 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) as previously described [27]. Briefly, cells were treated with or without H 2 O 2 and menadione for 2 h. The cells were subsequently harvested, washed with PBS, resuspended in PBS containing 20 µg/mL H 2 DCFDA, and incubated at 30 • C for 30 min. The fluorescence intensity was measured using an Accuri C6 flow cytometer (BD Biosciences).

Examination of Cell Morphology by Scanning Electron Microscopy (SEM)
To examine cell morphology, 6 × 10 7 cells were grown on polystyrene coverslips (Thermanox plastic coverslip 174950, Thermo Scientific) that were placed in each well of a 24-well microplate containing 1 mL SC medium with or without 30  the samples were dehydrated using serial ethanol solutions as previously described [22] and dried overnight in a 60 • C oven. Finally, the samples were examined and micrographs were collected using SEM S-4700, Type II (Hitachi, Minato-ku, Japan).

Measurement of Sod and Catalase Enzyme Activity
The Sod activity was measured using a Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (BioVision, Milpitas, CA) according to the manufacturer's instructions. Protein extraction was performed as described above in an extraction buffer containing 0.1 M Tris/HCl (pH 7.4), 0.5% Triton X-100, 5 mM β-mercaptoethanol, and 0.1 mg/mL phenylmethylsulfonyl fluoride. Proteins (2 µg in 250 µL solution) were mixed with 200 µL WST Solution and 20 µL SOD Enzyme Solution (BioVision) and incubated at 37 • C for 20 min. The absorbance at 450 nm was measured spectrophotometrically. The relative enzyme activity of Sod was calculated by the activity in the sfp1∆/sfp1∆ mutant divided by that in the wild-type strain.
Catalase enzyme activity was determined using a spectrophotometric method as previously described [28]. Briefly, 10 µg of protein was mixed with potassium phosphate buffer (75 mM, pH 7.0) and 10 mM H 2 O 2 to a volume of 1 mL. The rate of H 2 O 2 disappearance was detected by measuring the absorbance at 240 nm every 30 sec for a total of 2 min. One unit of catalase activity was defined as the amount of catalase required to degrade 1 µmole H 2 O 2 per min.
To measure the GSSG content, cells (in 100 µL protein extraction buffer) were mixed with 2 µL 2-vinylpyridine (2-VP). After the cells were disrupted, their supernatants were collected by centrifugation and incubated at room temperature for 1 h, allowing 2-VP to conjugate with GSH. Then, 6 µL triethanolamine was added to neutralize 2-VP, and the mixture was adjusted to pH 6~7. Finally, 20 µg of 2-VP treated proteins were incubated with 60 µL DTNB, 60 µL glutathione reductase, and 60 µL NADPH for 2 min, and the absorbance at 412 nm was measured. The GSH content was determined using the following formula:

Measurement of Glutathione Peroxidase Enzyme Activity
The glutathione peroxidase activity was determined using the glutathione reductase enzyme-coupling method as previously described [30] with some modifications. Proteins were extracted in an extraction buffer containing 50 mM potassium phosphate buffer (pH 7.2) with 5 mM EDTA. Briefly, 50 µL of protein was mixed with 10 µL NADPH (40 mM), 10 µL glutathione reductase (10 U/mL), and 10 µL glutathione (5 mM). Then, the mixture was added to 20 µL cumene hydroperoxide (0.25 mM) to initiate the reaction. The rate of NADPH oxidation was monitored by measuring the absorbance at 340 nm at a 1-min interval for 5 min. One unit of glutathione peroxidase activity was defined as the amount of enzyme that produced 1 µmol of GSSG/min.

Macrophage Killing and Phagocytosis Assay
The RAW264.7 mouse macrophage cell line was incubated with Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS) at 37 • C with 5% CO 2 . In the macrophage killing assay, C. albicans cells were cocultured with 10 6 RAW264.7 cells for 16 h at a multiplicity of infection (MOI) of 1:10. One milliliter of sterile ddH 2 O was added to promote macrophage lysis, C. albicans cells were collected by scraping from the bottom of each well and then spotted onto YPD agar plates, and colony-forming units (CFUs) were counted after incubation at 30 • C for 24 h.
In the phagocytosis assay, C. albicans cells were stained with 0.68 mg/mL fluorescein isothiocyanate (FITC) for 40 min and then cocultured with 2 × 10 6 cells of macrophages for 20 min at an MOI of 1. Then, 5 µg/mL calcofluor white was added to stain the nonphagocytosed C. albicans cells. The rate of phagocytosis was assessed using a fluorescence microscope (AIX0, Zeiss). Data were obtained from three independent experiments by analyzing at least 300 macrophages per well.

Statistical Analysis
Student's t-test was used to assess the statistical significance of differences in the wild-type strain versus the sfp1∆/sfp1∆ mutant. Statistical significance was indicated with a P-value <0.05.

Sfp1 is Involved in the C. albicans Response to Oxidative Stress
In our previous study, C. albicans Sfp1 was involved in cell adhesion and biofilm formation [22]. To reveal other functions of Sfp1, we used whole-genome DNA microarray to compare gene expression patterns between the sfp1-deleted (sfp1∆/sfp1∆) and wild-type strains. Among the 6,202 C. albicans genes that were evaluated, 2,365 genes exhibited a significant change in expression (P < 0.05) that was ≥1.5-fold. Based on C. albicans genome annotation (http://www.candidagenome.org), these genes are involved in a wide variety of biological processes ( Figure S1). Interestingly, a subset of genes involved in the oxidative stress response was upregulated in the sfp1∆/sfp1∆ mutant compared to their expression in the wild-type strain (Table 1), including GCS1 and GPX2, which encode gamma-glutamylcysteine synthetase and glutathione peroxidase, respectively. Moreover, the sfp1∆/sfp1∆ mutant also showed higher expression levels of genes encoding the components of oxidative stress signaling and regulation, including SSK1 and CAP1. Ssk1 is a response regulator of two-component system and functions upstream of the Hog1 mitogen-activated protein kinase (MAPK) to adapt cells to oxidative stress [32]. Cap1 is a transcription regulator that controls antioxidant gene expression [33,34]. To further investigate the functions of Sfp1 in the oxidative stress response, we determined the total antioxidative capacity of C. albicans. In Figure 1, the sfp1∆/sfp1∆ mutant showed an increase in antioxidative capacity compared to that in the wild-type and SFP1-reintegrated strains. Moreover, the total antioxidative capacity was largely enhanced in the sfp1∆/sfp1∆ mutant with hydrogen peroxide (H 2 O 2 )-induced oxidative stress. Therefore, the combined results of the DNA microarray and total antioxidant capacity assay suggested that C. albicans Sfp1 negatively regulates the oxidative stress response.   WT: wild-type strain; sfp1∆/sfp1∆: sfp1-deleted mutant; sfp1∆::SFP1/sfp1∆::SFP1: SFP1-reintegrated strain. The results are presented as the mean ± standard deviation (SD) of three independent experiments. * P < 0.05.

Sfp1 is Related to Cellular Susceptibility to Menadione/Superoxide
Superoxide is a primary ROS generated by phagocytes and several antifungals. To further understand the functions of Sfp1 in the oxidative stress response, we also determined the cellular response to the superoxide generator menadione. In a cell susceptibility assay, the sfp1∆/sfp1∆ mutant was more resistant to menadione than the wild-type and SFP1-reintegrated strains ( Figure 2A). Moreover, cell viability upon menadione treatment was also assessed through PI staining using flow cytometry. As shown in Figure 2B, the number of PI-positive cells was much lower in the sfp1∆/sfp1∆ mutant than in the wild-type and SFP1-reintegrated strains.
To further understand the correlation between Sfp1 and the cellular response to menadione-induced oxidative stress, intracellular ROS accumulation was detected. The cells were treated with sublethal doses of menadione and stained with the ROS indicator H 2 DCFDA. Intracellular ROS accumulation was then measured using flow cytometry. As shown in Figure 2C, the mean fluorescence intensity of the sfp1∆/sfp1∆ mutant was approximately 30-fold lower than those of the wild-type and SFP1-reintegrated cells. Together, these results indicate that the sfp1∆/sfp1∆ mutant is more resistant to menadione-induced oxidative stress and possesses a significantly lower intracellular ROS content upon the induction of superoxide than the other two tested strains. Superoxide is a primary ROS generated by phagocytes and several antifungals. To further understand the functions of Sfp1 in the oxidative stress response, we also determined the cellular response to the superoxide generator menadione. In a cell susceptibility assay, the sfp1Δ/sfp1Δ mutant was more resistant to menadione than the wild-type and SFP1-reintegrated strains ( Figure 2A). Moreover, cell viability upon menadione treatment was also assessed through PI staining using flow cytometry. As shown in Figure 2B, the number of PI-positive cells was much lower in the sfp1Δ/sfp1Δ mutant than in the wild-type and SFP1-reintegrated strains.
To further understand the correlation between Sfp1 and the cellular response to menadioneinduced oxidative stress, intracellular ROS accumulation was detected. The cells were treated with sublethal doses of menadione and stained with the ROS indicator H2DCFDA. Intracellular ROS accumulation was then measured using flow cytometry. As shown in Figure 2C, the mean fluorescence intensity of the sfp1Δ/sfp1Δ mutant was approximately 30-fold lower than those of the wild-type and SFP1-reintegrated cells. Together, these results indicate that the sfp1Δ/sfp1Δ mutant is more resistant to menadione-induced oxidative stress and possesses a significantly lower intracellular ROS content upon the induction of superoxide than the other two tested strains.

Sfp1 Affects SOD Gene Expression and Enzyme Activity
In C. albicans, superoxide is mainly detoxified by superoxide dismutases (Sods) that convert superoxide into the less toxic hydrogen peroxide [4,35]. Moreover, previous studies showed that Sods are involved in the C. albicans response to menadione, an ROS-generating antifungal, and macrophage killing [7,18,19]. Because the sfp1Δ/sfp1Δ mutant is resistant to menadione-induced oxidative stress (Figure 2A-C), we hypothesized that Sfp1 may regulate SOD expression. To test this

Sfp1 Affects SOD Gene Expression and Enzyme Activity
In C. albicans, superoxide is mainly detoxified by superoxide dismutases (Sods) that convert superoxide into the less toxic hydrogen peroxide [4,35]. Moreover, previous studies showed that Sods are involved in the C. albicans response to menadione, an ROS-generating antifungal, and macrophage killing [7,18,19]. Because the sfp1∆/sfp1∆ mutant is resistant to menadione-induced oxidative stress (Figure 2A-C), we hypothesized that Sfp1 may regulate SOD expression. To test this hypothesis, SOD gene expression levels and enzyme activity were compared among different C. albicans strains. Based on the results of real-time qPCR analysis, the expression of the SOD1, SOD4, and SOD5 genes, but not SOD2 and SOD3, was significantly upregulated in the sfp1∆/sfp1∆ mutant with menadione treatment compared to that in the other two strains ( Figure 3A and Figure S2). Finally, Sod enzyme activity was also measured. As shown in Figure 3B, the sfp1∆/sfp1∆ mutant exhibited 40% higher Sod enzyme activity than the wild-type and SFP1-reintegrated strains. These results further indicated that Sfp1 negatively regulates the cellular response to menadione/superoxide, possibly through its control of SOD gene expression and enzyme activity. hypothesis, SOD gene expression levels and enzyme activity were compared among different C. albicans strains. Based on the results of real-time qPCR analysis, the expression of the SOD1, SOD4, and SOD5 genes, but not SOD2 and SOD3, was significantly upregulated in the sfp1Δ/sfp1Δ mutant with menadione treatment compared to that in the other two strains ( Figure 3A and S2). Finally, Sod enzyme activity was also measured. As shown in Figure 3B, the sfp1Δ/sfp1Δ mutant exhibited 40% higher Sod enzyme activity than the wild-type and SFP1-reintegrated strains. These results further indicated that Sfp1 negatively regulates the cellular response to menadione/superoxide, possibly through its control of SOD gene expression and enzyme activity.

Sfp1 Is Also Related to Cellular Susceptibility to Hydrogen Peroxide
By the activity of Sods, superoxide is converted into H2O2. However, H2O2 is still toxic and highly reactive and requires further detoxification. Therefore, to explore the correlation between Sfp1 and H2O2 detoxification, cellular susceptibility to H2O2 was determined through PI staining and flow cytometry. The sfp1Δ/sfp1Δ mutant exhibited a much lower percentage of PI-positive cells after treatment with different concentrations of H2O2 than the wild-type and SFP1-reintegrated strains

Sfp1 Is Also Related to Cellular Susceptibility to Hydrogen Peroxide
By the activity of Sods, superoxide is converted into H 2 O 2 . However, H 2 O 2 is still toxic and highly reactive and requires further detoxification. Therefore, to explore the correlation between Sfp1 and H 2 O 2 detoxification, cellular susceptibility to H 2 O 2 was determined through PI staining and flow cytometry. The sfp1∆/sfp1∆ mutant exhibited a much lower percentage of PI-positive cells after treatment with different concentrations of H 2 O 2 than the wild-type and SFP1-reintegrated strains ( Figure 4A). For example, 55% of the wild-type cells were PI-positive following 90 mM H 2 O 2 treatment, whereas only 28% of the sfp1∆/sfp1∆ mutant were stained by PI ( Figure 4A). Moreover, the intracellular ROS content was measured in cells treated with a sublethal dose of H 2 O 2 . The sfp1∆/sfp1∆ mutant showed significantly lower fluorescence intensity in flow cytometric analysis following H 2 DCFDA staining than the wild-type and SFP1-reintegrated strains ( Figure 4B). Previous reports indicated that intracellular ROS accumulation is correlated with changes in the C. albicans cell surface, leading to a rough appearance with many protrusions and disc-like depressions [36,37]. Similar morphologies were observed when the wild-type strain was treated with H 2 O 2 ( Figure 4C). In contrast, the cell surfaces of the sfp1∆/sfp1∆ mutant (±H 2 O 2 ) and the wild-type (without H 2 O 2 treatment) remained relatively smooth ( Figure 4C). These results are consistent with the low level of intracellular ROS accumulation in the sfp1∆/sfp1∆ strain following H 2 O 2 treatment ( Figure 4B) and suggest that the sfp1∆/sfp1∆ mutant is better able to detoxify H 2 O 2 than the wild-type strain.
Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 20 ( Figure 4A). For example, 55% of the wild-type cells were PI-positive following 90 mM H2O2 treatment, whereas only 28% of the sfp1Δ/sfp1Δ mutant were stained by PI ( Figure 4A). Moreover, the intracellular ROS content was measured in cells treated with a sublethal dose of H2O2. The sfp1Δ/sfp1Δ mutant showed significantly lower fluorescence intensity in flow cytometric analysis following H2DCFDA staining than the wild-type and SFP1-reintegrated strains ( Figure 4B). Previous reports indicated that intracellular ROS accumulation is correlated with changes in the C. albicans cell surface, leading to a rough appearance with many protrusions and disc-like depressions [36,37]. Similar morphologies were observed when the wild-type strain was treated with H2O2 ( Figure  4C). In contrast, the cell surfaces of the sfp1Δ/sfp1Δ mutant (± H2O2) and the wild-type (without H2O2 treatment) remained relatively smooth ( Figure 4C). These results are consistent with the low level of intracellular ROS accumulation in the sfp1Δ/sfp1Δ strain following H2O2 treatment ( Figure 4B) and suggest that the sfp1Δ/sfp1Δ mutant is better able to detoxify H2O2 than the wild-type strain.

Sfp1 Regulates the Glutathione System to Detoxify Hydrogen Peroxide
Both catalase and the glutathione system participate in the conversion of H 2 O 2 into water [38,39] and provide overlapping defense against H 2 O 2 in the model yeast Saccharomyces cerevisiae [40]. Therefore, we determined whether the effect of Sfp1 on the cellular response to hydrogen peroxide is via catalase and/or the glutathione system. Interestingly, the sfp1∆/sfp1∆ mutant displayed slightly lower expression of the CAT1 catalase gene than the wild-type and SFP1-reintegrated strains ( Figure 5A). Moreover, catalase enzyme activity was lower in the sfp1∆/sfp1∆ mutant treated with H 2 O 2 than in the other tested strains ( Figure 5B). However, the sfp1∆/sfp1∆ mutant showed the upregulation of glutathione-related genes in DNA microarray and real-time qPCR analysis (Table 1 and Figure 6A). These genes included GCS1, GPX2, and GTT11. The GCS1, GPX2, and GTT11 genes encode gamma-glutamylcysteine synthetase, glutathione peroxidase, and glutathione S-transferase, respectively [20,41,42]. Therefore, the sfp1∆/sfp1∆ mutant exhibited lower catalase gene expression and enzyme activity but the increased expression of glutathione redox genes. These results suggest that the resistance to H 2 O 2 seen in the sfp1∆/sfp1∆ mutant is mainly due to changes in the glutathione redox system rather than catalase.
To further explore the link between Sfp1 and the glutathione redox system, a sodium selenite sensitivity assay was performed to detect alterations in cellular glutathione content. Glutathione is involved in selenite-induced oxidative stress and reacts with selenite to yield superoxide, causing cell death [43]. Therefore, cells containing a high level of glutathione are more sensitive to sodium selenite. Indeed, Figure 6B shows that the sfp1∆/sfp1∆ mutant was much more sensitive to sodium selenite than the wild-type strain, suggesting a higher cellular glutathione level in the mutant. Moreover, the total glutathione and GSH (reduced form of glutathione) content was also measured. The sfp1∆/sfp1∆ mutant contained a much higher total glutathione and GHS content than the wild-type and SFP1-reintegrated strains ( Figure 6C). Because GSH plays an important role in detoxifying ROS in Candida species [44], the high GSH content likely contributes to H 2 O 2 resistance in the sfp1∆/sfp1∆ mutant. Finally, glutathione peroxidases (Gpxs) catalyze the reduction of H 2 O 2 using GSH, and their activities were measured. As shown in Figure 6D, the sfp1∆/sfp1∆ mutant possessed much higher Gpx activity than the wild-type and SFP1-reintegrated strains. In summary, the deletion of SFP1 enhanced GCS1, GPX2, and GTT11 gene expression, which was correlated with a higher total glutathione and GSH content and high Gpx activity. These results suggest that Sfp1 is involved in the regulation of the glutathione redox system to detoxify H 2 O 2 . Both catalase and the glutathione system participate in the conversion of H2O2 into water [38,39] and provide overlapping defense against H2O2 in the model yeast Saccharomyces cerevisiae [40]. Therefore, we determined whether the effect of Sfp1 on the cellular response to hydrogen peroxide is via catalase and/or the glutathione system. Interestingly, the sfp1Δ/sfp1Δ mutant displayed slightly lower expression of the CAT1 catalase gene than the wild-type and SFP1-reintegrated strains ( Figure  5A). Moreover, catalase enzyme activity was lower in the sfp1Δ/sfp1Δ mutant treated with H2O2 than in the other tested strains ( Figure 5B). However, the sfp1Δ/sfp1Δ mutant showed the upregulation of glutathione-related genes in DNA microarray and real-time qPCR analysis (Table 1 and Figure 6A). These genes included GCS1, GPX2, and GTT11. The GCS1, GPX2, and GTT11 genes encode gammaglutamylcysteine synthetase, glutathione peroxidase, and glutathione S-transferase, respectively [20,41,42]. Therefore, the sfp1Δ/sfp1Δ mutant exhibited lower catalase gene expression and enzyme activity but the increased expression of glutathione redox genes. These results suggest that the resistance to H2O2 seen in the sfp1Δ/sfp1Δ mutant is mainly due to changes in the glutathione redox system rather than catalase.
To further explore the link between Sfp1 and the glutathione redox system, a sodium selenite sensitivity assay was performed to detect alterations in cellular glutathione content. Glutathione is involved in selenite-induced oxidative stress and reacts with selenite to yield superoxide, causing cell death [43]. Therefore, cells containing a high level of glutathione are more sensitive to sodium selenite. Indeed, Figure 6B shows that the sfp1Δ/sfp1Δ mutant was much more sensitive to sodium selenite than the wild-type strain, suggesting a higher cellular glutathione level in the mutant. Moreover, the total glutathione and GSH (reduced form of glutathione) content was also measured. The sfp1Δ/sfp1Δ mutant contained a much higher total glutathione and GHS content than the wildtype and SFP1-reintegrated strains ( Figure 6C). Because GSH plays an important role in detoxifying ROS in Candida species [44], the high GSH content likely contributes to H2O2 resistance in the sfp1Δ/sfp1Δ mutant. Finally, glutathione peroxidases (Gpxs) catalyze the reduction of H2O2 using GSH, and their activities were measured. As shown in Figure 6D, the sfp1Δ/sfp1Δ mutant possessed much higher Gpx activity than the wild-type and SFP1-reintegrated strains. In summary, the deletion of SFP1 enhanced GCS1, GPX2, and GTT11 gene expression, which was correlated with a higher total glutathione and GSH content and high Gpx activity. These results suggest that Sfp1 is involved in the regulation of the glutathione redox system to detoxify H2O2.

The Hog1 Signaling Pathway and the Transcription Factor Cap1 Are Related to the Sfp1-Mediated Oxidative Stress Response
The Hog1 MAPK pathway and Cap1-mediated transcriptional regulation are involved in the C. albicans oxidative stress response. Hog1 phosphorylation and Cap1 are required for the response of C. albicans to oxidants and phagocytic killing [17,45]. Based on our results that show Sfp1 is involved in the C. albicans oxidative stress response, we were interested in determining the relationship among Hog1, Cap1, and Sfp1.
As shown by DNA microarray analysis (Table 1) and real-time qPCR (Figure 7A), SSK1 that encodes a component of the Hog1 signaling pathway, was upregulated in the sfp1∆/sfp1∆ mutant compared to their expression in the other tested strains. In particular, C. albicans utilizes Ssk1 to adapt cells to oxidative stress [32]. As shown by Western blotting in Figure 7B, the sfp1∆/sfp1∆ mutant contained a significant amount of phosphorylated Hog1 at time point zero, and Hog1 phosphorylation was strongly enhanced in cells treated with H 2 O 2 for 15 and 30 min ( Figure 7B). However, as shown by a longer exposure of blot, phosphorylated Hog1 was detected in the wild-type strain in cells treated with H 2 O 2 ( Figure S3). These results suggest that the Sfp1-mediated oxidative stress response involves the Hog1 signaling pathway.
Microorganisms 2019, 7, x FOR PEER REVIEW 12 of 20 < 0.001. (D) Cells were treated with 1 mM H2O2 for 2 h. The enzyme activity of glutathione peroxidase (Gpx) was determined by the oxidation of NADPH. The results are presented as the mean ± standard deviation (SD) of two independent experiments. * P < 0.05.

The Hog1 Signaling Pathway and the Transcription Factor Cap1 Are Related to the Sfp1-Mediated Oxidative Stress Response
The Hog1 MAPK pathway and Cap1-mediated transcriptional regulation are involved in the C. albicans oxidative stress response. Hog1 phosphorylation and Cap1 are required for the response of C. albicans to oxidants and phagocytic killing [17,45]. Based on our results that show Sfp1 is involved in the C. albicans oxidative stress response, we were interested in determining the relationship among Hog1, Cap1, and Sfp1.
As shown by DNA microarray analysis (Table 1) and real-time qPCR (Figure 7A), SSK1 that encodes a component of the Hog1 signaling pathway, was upregulated in the sfp1Δ/sfp1Δ mutant compared to their expression in the other tested strains. In particular, C. albicans utilizes Ssk1 to adapt cells to oxidative stress [32]. As shown by Western blotting in Figure 7B, the sfp1Δ/sfp1Δ mutant contained a significant amount of phosphorylated Hog1 at time point zero, and Hog1 phosphorylation was strongly enhanced in cells treated with H2O2 for 15 and 30 min ( Figure 7B). However, as shown by a longer exposure of blot, phosphorylated Hog1 was detected in the wildtype strain in cells treated with H2O2 ( Figure S3). These results suggest that the Sfp1-mediated oxidative stress response involves the Hog1 signaling pathway.

The sfp1∆/sfp1∆ Mutant is Resistant to Macrophage Killing
During infection, C. albicans encounters host phagocytes that produce ROS to kill the pathogen. Due to the involvement of Sfp1 in the oxidative stress response, the interaction between the macrophage cell line RAW264.7 and C. albicans was investigated. There was no statistically significant difference in phagocytosis between the sfp1∆/sfp1∆ mutant, wild-type, and SFP1-reintegrated strains ( Figure 8A). However, the wild type and SFP1-reintegrated strains were relatively sensitive to macrophage killing and displayed~30% viability ( Figure 8B). In contrast, the sfp1∆/sfp1∆ mutant was extremely resistant to macrophage killing ( Figure 8B). Notably, growth of the sfp1∆/sfp1∆ mutant was even enhanced in the macrophage.

The sfp1Δ/sfp1Δ Mutant is Resistant to Macrophage Killing
During infection, C. albicans encounters host phagocytes that produce ROS to kill the pathogen. Due to the involvement of Sfp1 in the oxidative stress response, the interaction between the macrophage cell line RAW264.7 and C. albicans was investigated. There was no statistically significant difference in phagocytosis between the sfp1Δ/sfp1Δ mutant, wild-type, and SFP1-reintegrated strains ( Figure 8A). However, the wild type and SFP1-reintegrated strains were relatively sensitive to macrophage killing and displayed ~30% viability ( Figure 8B). In contrast, the sfp1Δ/sfp1Δ mutant was extremely resistant to macrophage killing ( Figure 8B). Notably, growth of the sfp1Δ/sfp1Δ mutant was even enhanced in the macrophage.

The sfp1Δ/sfp1Δ Mutant is Resistant to ROS-Generating Antifungals
Miconazole and caspofungin are commonly used antifungals that induce ROS to kill C. albicans [7,8]. In addition, the addition of antioxidants impairs ROS-generating antifungal efficacy [7,21]. Because the sfp1Δ/sfp1Δ mutant exhibits high antioxidative activity, we were interested in linking the susceptibility of the sfp1Δ/sfp1Δ mutant to ROS-generating antifungals. As shown in Figure 9A, the result of spot assay showed that the sfp1Δ/sfp1Δ mutant was resistant to miconazole and caspofungin, as opposed to the controls. Moreover, H2DCFDA staining was performed to measure the intracellular ROS content upon antifungal drug treatment. The sfp1Δ/sfp1Δ mutant exhibited less ROS accumulation with miconazole and caspofungin treatment than the controls ( Figure 9B).

The sfp1∆/sfp1∆ Mutant is Resistant to ROS-Generating Antifungals
Miconazole and caspofungin are commonly used antifungals that induce ROS to kill C. albicans [7,8]. In addition, the addition of antioxidants impairs ROS-generating antifungal efficacy [7,21]. Because the sfp1∆/sfp1∆ mutant exhibits high antioxidative activity, we were interested in linking the susceptibility of the sfp1∆/sfp1∆ mutant to ROS-generating antifungals. As shown in Figure 9A, the result of spot assay showed that the sfp1∆/sfp1∆ mutant was resistant to miconazole and caspofungin, as opposed to the controls. Moreover, H 2 DCFDA staining was performed to measure the intracellular ROS content upon antifungal drug treatment. The sfp1∆/sfp1∆ mutant exhibited less ROS accumulation with miconazole and caspofungin treatment than the controls ( Figure 9B).

Discussion
C. albicans is challenged by oxidative stress from host phagocytes and antifungals [3,4]. C. albicans has complex antioxidant systems, signaling pathways, and transcriptional regulatory machinery to cope with oxidative stress. One key mechanism known to activate the expression of antioxidant genes is primarily mediated by Cap1, a bZIP transcription factor in the AP-1 family [47,48]. After exposure to H2O2, Cap1 is activated by the oxidation of its redox-active cysteine residues, allowing the nuclear accumulation of Cap1 [21]. Within the nucleus, Cap1 is phosphorylated and induces the expression of many genes, including CAT1, which encodes catalase, and TRX1, which encodes thioredoxin [46]. Moreover, C. albicans cells lacking CAP1 are sensitive to ROS and phagocyte killing [34,49]. Another mechanism related to the C. albicans oxidative response is Hog1 MAPK signaling [50]. Hog1 is activated in C. albicans in response to diverse stimuli, such as high doses of H2O2, which results in its nuclear accumulation [14]. Moreover, global transcriptional analysis using DNA microarray revealed that 46 core stress genes induced in response to H2O2 are Hog1-dependent [51].
In this study, we demonstrated that the transcription factor Sfp1 is also involved in the oxidative stress response of C. albicans. We showed that the sfp1Δ/sfp1Δ mutant possesses a higher total antioxidant capacity, Sod enzyme activity, GSH content, and glutathione peroxidase activity than the wild-type and SFP1-reintegrated strains (Figure 1, 3, and 6). In addition, the sfp1Δ/sfp1Δ mutant was more resistant to phagocyte killing and ROS-inducing antifungals (Figure 8 and 9). Moreover, DNA microarray analysis and real-time qPCR revealed that the expression of many oxidative stress response-related genes was upregulated in the sfp1Δ/sfp1Δ mutant compared to their expression in the other two strains, including CAP1 (Table 1 and Figure 7A). Moreover, many oxidative stress Figure 9. The effect of SFP1 deletion on cellular susceptibility to ROS-generating antifungals and the accumulation of intracellular ROS upon antifungal treatment. (A) One colony was inoculated into YPD medium and grown at 30 • C overnight. This culture was harvested by centrifugation and washed with sterile double-distilled water (ddH 2 O). Cells were ten-fold serially diluted and spotted onto YPD agar with or without 8 µg/mL of an antifungal. The agar plates were incubated at 30 • C for 3-4 days. Representative images of three independent experiments with identical results are shown. (B) Cells were treated with 3 µg/mL miconazole or 1 µg/mL caspofungin for 2 h. Intracellular ROS were measured using H 2 DCFDA staining and quantified by a flow cytometer. The results are presented as the mean ± standard deviation (SD) of three independent experiments. ** P < 0.01.

Discussion
C. albicans is challenged by oxidative stress from host phagocytes and antifungals [3,4]. C. albicans has complex antioxidant systems, signaling pathways, and transcriptional regulatory machinery to cope with oxidative stress. One key mechanism known to activate the expression of antioxidant genes is primarily mediated by Cap1, a bZIP transcription factor in the AP-1 family [47,48]. After exposure to H 2 O 2 , Cap1 is activated by the oxidation of its redox-active cysteine residues, allowing the nuclear accumulation of Cap1 [21]. Within the nucleus, Cap1 is phosphorylated and induces the expression of many genes, including CAT1, which encodes catalase, and TRX1, which encodes thioredoxin [46]. Moreover, C. albicans cells lacking CAP1 are sensitive to ROS and phagocyte killing [34,49]. Another mechanism related to the C. albicans oxidative response is Hog1 MAPK signaling [50]. Hog1 is activated in C. albicans in response to diverse stimuli, such as high doses of H 2 O 2 , which results in its nuclear accumulation [14]. Moreover, global transcriptional analysis using DNA microarray revealed that 46 core stress genes induced in response to H 2 O 2 are Hog1-dependent [51].
In this study, we demonstrated that the transcription factor Sfp1 is also involved in the oxidative stress response of C. albicans. We showed that the sfp1∆/sfp1∆ mutant possesses a higher total antioxidant capacity, Sod enzyme activity, GSH content, and glutathione peroxidase activity than the wild-type and SFP1-reintegrated strains (Figure 1, Figure 3, and Figure 6). In addition, the sfp1∆/sfp1∆ mutant was more resistant to phagocyte killing and ROS-inducing antifungals (Figures 8 and 9). Moreover, DNA microarray analysis and real-time qPCR revealed that the expression of many oxidative stress response-related genes was upregulated in the sfp1∆/sfp1∆ mutant compared to their expression in the other two strains, including CAP1 (Table 1 and Figure 7A). Moreover, many oxidative stress response genes (e.g., SOD1, GCS1, GTT11, CIP1, EBP1, IFD6, and OYE32) were regulated by both Sfp1 and Cap1 [ [46,47] and Table 1]. Recently, Sfp1 was found to reciprocally modulate carbon source-conditional stress adaptation with another transcription factor, Rtg3 [52]. Sfp1 regulates oxidative stress response genes in a carbon source-dependent manner [52]. In the study of Kastora et al. [52], the sfp1∆/sfp1∆ mutant was more sensitive to H 2 O 2 than the wild-type strain. These results contrasted with the increased sensitivity of the sfp1∆/sfp1∆ mutant to H 2 O 2 in this study (Figure 1). Our explanation for this result is that these two studies use C. albicans strains from a different genetic background and different concentrations of H 2 O 2 . Taken together, our and other studies highlight the complex transcription regulation network of the oxidative stress response. However, the epistatic relationship between Sfp1, Rtg3, and Cap1 needs to be further investigated by either construction of a double mutant or chromatin immunoprecipitation to determine the possible interaction between the Sfp1 protein and the CAP1 promoter. Additionally, whether Sfp1 directly controls oxidative stress genes also requires further study.
In addition to Cap1, Sfp1 is also associated with the Hog1 MAPK signaling pathway. As shown in Figure 7A, the sfp1∆/sfp1∆ mutant exhibited increased gene expression of the response regulator SSK1, which is an upstream component of the Hog1 cascade, compared to its expression in the other tested strains [53]. In particular, Ssk1 is required for oxidative stress response [32,54], phagocyte killing, and virulence in a disseminate murine model of candidiasis [55,56]. Moreover, Hog1 phosphorylation was enhanced in the sfp1∆/sfp1∆ mutant compared to that in the wild-type strain in the absence and presence of H 2 O 2 ( Figure 7). Finally, compared to the catalase Cat1, the glutathione system seems to play a greater role in H 2 O 2 detoxification in the sfp1∆/sfp1∆ mutant (Figures 5 and 6). Glutathione is the most important thiol-containing molecule required to maintain the redox homeostasis, as it functions as redox buffer, antioxidant, and enzyme cofactor against oxidative stress [57][58][59]. Interestingly, the sfp1∆/sfp1∆ mutant exhibited the upregulation of the MET1 and CYS3 genes (Table 1), which encode enzymes involved in methionine and cysteine biosynthesis, respectively. Methionine and cysteine are the precursors of glutathione biosynthesis [42]. Recently, the relationship between Hog1 and Sfp1 was revealed, in which Hog1 is required for Sfp1-dependent ribosome biogenesis (RiBi) gene expression and recruitment to target promoters [60]. However, future studies to examine the role of Hog1 and Sfp1 in C. albicans amino acid and glutathione biosynthesis are still needed.
In addition to C. albicans, transcription factors that are vital for controlling oxidative stress response have been also studied in other fungal species [61][62][63][64]. For example, Skn7 and Yap1 are the AP-1-like bZIP transcription factors in S. cerevisiae. Yap1 is the orthologue of C. albicans Cap1 and accumulates in the nucleus following exposure to H 2 O 2 [62]. Previous studies indicated that Yap1 collaborates with Skn7 to control many oxidative stress response genes [62,[65][66][67]. Similarly, Candida glabrata Yap1 and Skn7 are involved in oxidative stress response by cooperatively binding to the upstream region of core oxidative stress genes [68]. Moreover, Ada2 is suggested to orchestrate C. glabrata against ROS-mediated immune defenses during infection [69]. Evidence for Skn7 having a role in virulence is also reported in different fungal species [70]. Therefore, oxidative stress adaptation is not only essential for cell survival, but also an important virulence trait. In this study, our results showed the multiple functions of Sfp1 and the regulatory complexity of the C. albicans oxidative stress response. These results should also provide useful insight into the oxidative stress response in other important human fungal pathogens.