The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress

ABSTRACT The fungal pathogen Candida albicans is capable of causing lethal infections in humans. Its pathogenic potential is due in part to the ability to resist various stress conditions in the host, including oxidative stress. Recent studies showed that a family of four flavodoxin-like proteins (Pst1, Pst2, Pst3, Ycp4) that function as quinone reductases promotes resistance to oxidation and is needed for virulence. Therefore, in this study, Zta1 was examined because it belongs to a structurally distinct family of quinone reductases that are highly conserved in eukaryotes and have been called the ζ-crystallins. The levels of Zta1 in C. albicans rapidly increased after exposure to oxidants, consistent with a role in resisting oxidative stress. Accumulation of reactive oxygen species (ROS) was significantly higher in cells lacking ZTA1 upon exposure to quinones and other oxidants. Furthermore, the deletion of ZTA1 in a mutant lacking the four flavodoxin-like proteins, resulted in further increased susceptibility to quinones, indicating that these distinct quinone reductases work in combination. These results demonstrate that Zta1 contributes to C. albicans survival after exposure to oxidative conditions, which increases the understanding of how C. albicans resist stressful conditions in the host. IMPORTANCE Candida albicans is an important human pathogen that can cause lethal systemic infections. The ability of C. albicans to colonize and establish infections is closely tied to its highly adaptable nature and capacity to resist various types of stress, including oxidative stress. Previous studies showed that four C. albicans proteins belonging to the flavodoxin-like protein family of quinone reductases are needed for resistance to quinones and virulence. Therefore, in this study, we examined the role of a distinct type of quinone reductase, Zta1, and found that it acts in conjunction with the flavodoxin-like proteins to protect against oxidative stress.

oxidative stress, preventing lipid peroxidation, and for virulence in mice (7,8).The FLPs belong to a large family of NADPH:quinone oxidoreductases that are present in many organisms, including bacteria, fungi, and plants, but are not conserved in mammals (9).FLPs are enriched in plasma membrane eisosome domains where they are thought to protect the C. albicans plasma membrane by reducing ubiquinone to ubiquinol so that it can reduce ROS (7).FLPs are also important for resisting oxidative stress caused by the treatment of C. albicans with a variety of smaller quinone molecules (7,8).FLPs are thought to therefore play a critical role in protecting cells from quinones that are created as secondary metabolites, or in many cases are used by plants and insects as defense molecules (10)(11)(12).Due to their redox-active nature, quinones can cause oxidative stress by undergoing cycles of oxidation and reduction, which can consume reducing agents such as glutathione and NADPH.In addition, FLPs carry out a two-electron reduction of quinones, which prevents the formation of dangerous semiquinone radicals that interact with oxygen to form superoxide anion radicals, leading to cell damage (10,11).
In addition to FLPs, many organisms contain members of a distinct group of quinone oxidoreductases (QORs) that belong to the medium-chain dehydrogenase/reductase (MDR) superfamily (13,14).The first QOR of this group to be described was the mammalian ζ-crystallin, a major protein of the eye lens that was observed to also be present in other cell types where it is capable of reducing quinones using NADPH as a cofactor (15)(16)(17)(18).However, unlike the FLPs, it is believed that these enzymes catalyze a one-electron reduction of quinones (16).A ζ-crystallin homologous protein called Zta1 has been described in Saccharomyces cerevisiae and Pichia pastoris, and it has been suggested that it acts as a NADPH:quinone oxidoreductase that protects cells from oxidative stress (17,18).However, little is known about it since data are limited for S. cerevisiae Zta1 and C. albicans Zta1 has not been studied previously.Therefore, in this study, we examined the production of Zta1 and its function in protecting C. albicans from oxidative stress.In addition, a mutant lacking ZTA1 as well as the four flavodoxin-like proteins (zta1Δ pst1Δ pst2Δ pst3Δ ycp4Δ) was constructed to determine the phenotype of a cell lacking all five QOR genes.These data indicate that Zta1 acts in combination with FLPs to protect C. albicans from oxidative stress.

Zta1 proteins are highly conserved between C. albicans and other fungi
The medium-chain dehydrogenase/reductase family includes the QOR known as ζ-crystallin that was first discovered in the lenses of camels and guinea pigs but was subsequently found in a wide range of eukaryotic cells (19).Interestingly, a protein with homology to ζ-crystallin was identified in fungi.The crystal structure of the S. cerevisiae ζ-crystallin, termed Zta1, has been described and shows that it is structurally similar to mammalian ζ-crystallin proteins (20).Comparison of the Zta1 proteins from S. cerevisiae and C. albicans revealed a high degree of sequence identity (56.8%) and there was 100% conservation of residues implicated in catalytic activity (Fig. 1).Although the human homolog Cryz has a low overall sequence identity to Zta1 (~26%), the active site residues are mostly conserved.Therefore, C. albicans Zta1 belongs to the highly conserved family of ζ-crystallin proteins.

Zta1 is localized to the cytoplasm and induced by oxidation
To examine the subcellular localization and production of Zta1, a triple GFP tag (3×GFP) was fused to the 3′ end of the ZTA1 open reading frame.Analysis of log-phase cells by fluorescence microscopy revealed that Zta1-3×GFP localized to the cytoplasm.The basal level of Zta1-3×GFP was low in cells grown in standard medium, but the signal was induced by treatment with several different quinones including p-benzoquinone (BZQ) (4-fold), 2-tert-butyl-1,4-benzoquinone (TBBQ) (1.6-fold), and menadione (MEN) (2.5-fold).(Fig. 2A and D).Interestingly, hydrogen peroxide (H 2 O 2 ) also significantly increased Zta1 production (3.5-fold), indicating that ZTA1 expression is induced by oxidative stress, not just by quinones.Concentrations as low as 10 µM BZQ were sufficient to achieve a threefold increase in Zta1-3×GFP production (Fig. 2B and E).Treatment with 100 µM BZQ resulted in a reduction of Zta1-3×GFP induction, probably due to toxicity.ZTA1 was induced quickly after exposure to BZQ.The relative level of Zta1-3×GFP increased by a 2-fold and 3.1-fold increase after 30 and 60 minutes, respectively (Fig. 2C and F).
Since Zta1-3×GFP was localized to the cytoplasm, western blot analysis was carried out to determine whether the expected fusion protein was produced or if free GFP was being proteolytically clipped off.A band of ~118 kDa was detected with anti-GFP antibodies, indicating the full-length Zta1-3×GFP fusion protein was produced.Only a weak signal was detected at the expected position for free GFP (~26.7 kDa), confirming that Zta1-3×GFP is cytoplasmic.

Zta1 prevents the accumulation of ROS
The ability of Zta1 to protect against ROS was examined using the fluorescent probe H 2 DCFDA, which has been previously used to quantify ROS accumulation in Candida cells (21).Wild type and zta1Δ/Δ mutant cells treated with 30 µM of the quinones BZQ or TBBQ did not lead to detectable accumulation of ROS using H 2 DCFDA (Fig. 4A and  D).However, both quinones significantly induced ROS accumulation at 100 µM in the zta1Δ/Δ mutant, which was abolished in the complemented strain zta1+ (Fig. 4B, C, E,  and F).The median fluorescence intensity of zta1Δ/Δ cells that were incubated with TBBQ was significantly higher when compared with cells exposed to BZQ (t-test P = 0.021).These data indicate that Zta1 can reduce ROS accumulation caused by quinones and protect C. albicans from oxidative damage and that TBBQ induces higher ROS production than does BZQ in the zta1Δ/Δ cells.****, P ≤ 0.0001, by one-way analysis of variance (ANOVA).○, P ≤ 0.05, by t-test (100 µM of p-benzoquinone vs 100 µM 2-tert-butyl-1,4-benzoquinone).The wild-type control strain was LLF100.The strains are described in Table 1.

Zta1 acts in combination with the FLPs to protect C. albicans from quinones
To determine whether Zta1 is important for C. albicans resistance to quinones, we compared a wild-type C. albicans control strain with the zta1Δ/Δ mutant and the complemented strain zta1+.However, no differences in sensitivity were detected with disk diffusion halo assays when the strains were exposed to BZQ, MEN, or TBBQ.Since C. albicans also possess QORs that belong to the FLP family, we tested whether ZTA1 might be more important in the absence of the FLPs.ZTA1 was deleted from a quadruple mutant lacking all four FLPs (pst1Δ/Δ pst2Δ/Δ pst3Δ/Δ ycp4Δ/Δ; referred to as Δ/Δ/Δ/Δ for simplicity) (7) to create a quintuple mutant lacking the four FLPs and ZTA1 (referred to as Q Mut).We also generated a Q mutant complemented with ZTA1 (pst1Δ/Δ pst2Δ/Δ pst3Δ/Δ ycp4Δ/Δ zta1Δ/Δ + ZTA1; referred to as Q Mut+).Although no difference was observed when cells were treated with BZQ or MEN, the Q Mut was more sensitive to killing by TBBQ when compared with the Δ/Δ/Δ/Δ mutant, and this sensitivity was partially reverted in the Q Mut+ complemented with ZTA1 (Fig. 5).Perhaps TBBQ is more effective at killing C. albicans because it is more non-polar than the other quinones used in this study and may therefore cross membranes more efficiently.These data indicate that Zta1 synergizes with the FLPs to detoxify certain quinones.

Zta1 protection appears to be specific to quinones
To determine if Zta1 protects C. albicans against other sources of oxidative stress, the susceptibility of cells to other oxidizing agents was examined including H 2 O 2 , tert-butylhydroperoxide (Fig. 6), and the thiol oxidizing compound diamide (data not shown).
Neither zta1Δ/Δ nor the Q Mut strain showed increased susceptibility to these other oxidizing agents (Fig. 6).Thus, although Zta1 is induced by H 2 O 2 , it does not appear to play a major role in protecting against this type of oxidative stress.

zta1Δ/Δ shows a trend of increased susceptibility to killing by neutrophils
To assess the potential significance of ZTA1 in C. albicans's resistance to attack by the immune system, we analyzed the ability of human neutrophils to kill C. albicans wild-type and mutant strains.Notably, increased vulnerability of zta1Δ/Δ cells to neutrophil-medi ated killing was observed, which was reversed in the ZTA1 complemented strain (Fig. 7).
Unfortunately, due to variation in the data, this trend did not reach statistical significance when analyzed by one-way analysis of variance (ANOVA) or t-test.Donor-dependent variations when working with neutrophils are known to occur since environmental aspects such as temperature, pH, oxygen and glucose levels can have a strong influence on the function of neutrophils (22).Deletion of the ZTA1 gene in the Δ/Δ/Δ/Δ mutant did not yield an obvious increase in susceptibility to neutrophils; similar survival levels were observed between the zta1Δ/Δ, Δ/Δ/Δ/Δ, Q mut, and Q mut+ (complemented with ZTA1).

ZTA1 role in C. albicans virulence
The role of ZTA1 in C. albicans virulence was investigated in a mouse model of hemato genously disseminated candidiasis (23).The wild-type, Δ/Δ/Δ/Δ, Q Mut, and Q Mut+ strains were used to infect BALB/c mice via tail vein injection with 2.5 × 10 5 C. albicans cells.Since it has been shown that the Δ/Δ/Δ/Δ mutant is non-virulent and is cleared after ~4 days of infection (7), we focused on measuring the colony-forming units in the kidney (CFU/g kidney) after 2 and 3 days of infection.The kidneys are an established target organ for testing the capacity of C. albicans to cause an infection since growth occurs rapidly in the kidneys during the first 2 days after injection (24).Interestingly, at day 2 (Fig. 8A) and day 3 (Fig. 8B) post-infection, the Q Mut strain showed lower CFU/g kidney than did the Δ/Δ/Δ/Δ strain, suggesting a role for ZTA1 in virulence.In contrast, the Q Mut+ strain had a similar fungal burden as the Δ/Δ/Δ/Δ, indicating this effect was reversed by reintroducing the ZTA1 gene.However, this trend in the results did not reach statistical significance when analyzed by ANOVA or t-test.Nonetheless, the Δ/Δ/Δ/Δ and Q Mut strains had lower kidney fungal burden than the wild-type control strain, highlighting the importance of quinone reductases in virulence.Although statistical significance was lacking, the overall trends of these studies suggest that Zta1 contributes to C. albicans virulence.

DISCUSSION
Fungal cells face various challenges when colonizing a host, including ROS that can cause widespread damage to proteins, lipids, and nucleic acids (25).Among the arsenal employed by fungal cells to deal with this stress are QOR enzymes capable of reducing quinones into hydroquinones (13).This mechanism holds significance as quinones have the potential to alkylate proteins and DNA or undergo autoxidation, resulting in the production of semiquinone radicals and the generation of ROS (9,11,26,27).Further more, QORs can also reduce ubiquinone, a well-known component of the mitochondrial membrane that participates in cellular respiration, but is also present in other mem branes, including the plasma membrane, where it functions as an antioxidant (28).In fact, recent studies revealed that four members of the FLP family of flavin-dependent NADPH:quinone oxidoreductases (Pst1, Pst2, Pst3, Ycp4) are needed for resistance to quinones and virulence (7).The FLP family members in C. albicans are thought to act in part by catalyzing a two-electron reduction of ubiquinone that enables it to then reduce ROS in the plasma membrane (7).The FLPs are localized to specialized plasma membrane domains called eisosomes, which are thought to facilitate a role in protecting the C. albicans plasma membrane polyunsaturated fatty acids (PUFAs) which are more prone to lipid peroxidation.This type of lipid oxidation can trigger a chain reaction that spreads to other PUFAs and then the peroxidized lipids can damage proteins and DNA (8,29).Another QOR present in fungi is the ζ-crystallin-like protein Zta1, which belongs to a structurally distinct family that is believed to catalyze a one-electron reduction of quinones (16,18).Due to the FLP's importance, we decided to investigate the role of Zta1 in C. albicans.The high degree of amino acid conservation in the active site of C. albicans Zta1 strongly indicates that it functions as a QOR, as in S. cerevisiae (Fig. 1).Supporting this notion, C. albicans Zta1 was rapidly induced upon exposure to p-benzoquinone, 2-tert-butyl-1,4-benzoquinone, menadione, and hydrogen peroxide (Fig. 2 and 3).However, despite its induction by H 2 O 2 , Zta1 does not appear to play a significant role in protecting cells against H 2 O 2 or other oxidants such as tert-butyl-hydroperoxide (Fig. 6) and diamide (data not shown).Previous studies have reported the upregulation of flavin-containing QORs under certain conditions, such as increased temperature (26).Moreover, Zta1 production in S. cerevisiae increased following treatment with rapamy cin, heat shock, and during the stationary phase, when cells are starved and toxic compounds accumulate (30).These observations suggest that Zta1 may be part of a more general fungal cellular response to stress, which could explain its induction by H 2 O 2 while not directly providing protection against it.
Assays utilizing H 2 DCFDA revealed that the zta1Δ/Δ exhibited increased accumula tion of ROS upon exposure to quinones, indicating it protects against this type of oxidative stress (Fig. 4).Surprisingly, we did not observe increased cell death of the zta1Δ/Δ mutant in response to quinones, despite a previous report that the S. cerevisiae zta1Δ mutant showed slightly increased susceptibility to menadione (18).However, the Q Mut, which lacks all four FLPs (pst1Δ/pst2Δ/pst3Δ/ycp4Δ) as well as ZTA1, exhibited greater susceptibility to TBBQ, although not to BZQ or MEN.One possibility for this specific susceptibility is that TBBQ, being the most non-polar quinone tested in our study (8), may cross the plasma membrane more readily and enter the cytoplasm where Zta1 is located (Fig. 2).In this scenario, C. albicans FLPs may preferentially act at the plasma membrane where they are localized and Zta1 may be important to reduce TBBQ in the cytoplasm, where it was detected.
To determine if ZTA1 is important for C. albicans to avoid attack by the immune system, the zta1ΔΔ mutant was assessed for its ability to survive incubation with human neutrophils.The results showed a trend of increased killing of the zta1ΔΔ mutant by neutrophils, which was reversed by reintroduction of the ZTA1 gene in the complemen ted strain (Fig. 7).The presence or absence of ZTA1 in cells lacking all four FLPs did not appear to obviously impact in C. albicans survival, perhaps because cells lacking the FLPs are already more susceptible to neutrophils.1.
Previous studies demonstrated that the FLPs are critical for C. albicans infection in mice, as a mutant lacking all four FLPs was avirulent and cleared from the kidney at early times after infection correlated with the influx of neutrophils (7).Considering the limited role of zta1Δ/Δ in susceptibility to quinones in vitro, we decided to investigate whether zta1Δ/Δ would exacerbate the previously described virulence defect of the FLP mutant (pst1Δ/Δ, pst2Δ/Δ, pst3Δ/Δ, ycp4Δ/Δ) (7) by assessing the kidney fungal burden in infected mice.Notably, we observed a trend toward reduced CFU/g kidney of the Q Mut at both day 2 and day 3 post-infection.Complementation of Q Mut with ZTA1 restored its clearance to levels comparable to those of the FLP mutant.This finding aligns with studies implicating QORs in the virulence of other organisms, such as Xanthomonas citri, Mycobacterium turberculosis, and Staphylococcus aureus (31)(32)(33).This suggests that Zta1 contributes, at least partially, to C. albicans ability to resist in the host and evade its immune system.
Collectively, our data indicate that Zta1 is rapidly induced by quinones and protects C. albicans against quinone-induced damage.It also protects against the accumulation of ROS caused by quinones and might contribute to C. albicans capacity to establish an infection.Zta1 function seems to overlap with other QORs, such as the FLPs, and due to their difference in localization, they seem to complement each other.These  1).CFU/g of the kidney was determined at (A) day 2 and (B) day 3 post-infection.Four mice were injected with each strain for each day of analysis and their mean is presented.results highlight the important role of QORs in C. albicans repertoire of strategies to be a successful pathogen.

Strains and media
C. albicans strains used are listed in Table 1.Strains were kept in YPD (1% yeast extract, 2% peptone, 2% glucose) agar plates.Prior to experiments, cultures were grown in YPD medium (2% dextrose, 1% peptone, 2% yeast extract, 80 mg/L uridine) (34).A 3×GFPγ tag was fused to the 3′ end of the open reading frame of ZTA1 by homologous recombination as previously described (35,36).The DNA was introduced into C. albicans cells by electroporation and allowed to recombine with the homologous regions of the ZTA1 gene.Strains were verified by PCR analysis and microscopic examination of GFPγ fluorescence in a Zeiss Axiovert 200M microscope equipped with an AxioCam HRm camera and Zeiss ZEN software.Mutant strains of zta1 were generated using a transient expression of CRISPR-Cas9 to obtain homozygous deletion of the target gene, as previously described (37).A 20 bp sequence of the sgRNA was used to target CRISPR-Cas9 to ZTA1 in strain SN152.The cells were also transformed with a healing fragment that was constructed by using PCR to amplify the HIS1 selectable marker with primers that included 80 bases of homology to the sequences flanking ZTA1.Strains in which ZTA1 was deleted and replaced with HIS1 were then selected on a medium lacking histidine.To create the Q Mut, the same CRISPR-Cas9 method was used to delete ZTA1 and replace it with an ARG4 selectable marker.To complement the zta1Δ/Δ strains, a copy of the wild-type ZTA1 gene sequence was integrated into the NEUT5L region of the genome using a plasmid vector created by the gap-repair method, as previously described (38).The PCR fragment was constructed by amplification of the genomic DNA from 0.5 kb upstream of the start codon and 0.25 kb downstream of the stop codon of the ZTA1 gene from C. albicans SC5314 using primers 5227 and 5228, which includes 20 bp homology to the ZTA1 upstream and downstream regions and 40 bp homology with the plasmid pDIS3, that carries the NAT1 resistance cassette.The PCR fragment was recombined into SmaI-digested pDIS3 in S. cerevisiae strain L40, generating the pDIS3-ZTA1-NAT1 construct, which was subsequently released from the pDIS3 vector by SfiI digestion and transformed into the zta1Δ/Δ or Q Mut strain by electroporation.The primers used are listed in Table 2.

Sequence alignments of Zta1 proteins
Zta1 protein sequence was obtained from the S. cerevisiae Genome Database (http://www.yeastgenome.org).Basic Local Alignment Search Tool (BLAST) searches were carried out to identify Zta1 homologs in the C. albicans Genome Database (http://www.candidagenome.org)and genome sequences present at the website of the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov/BLAST/).Multiple sequence alignments of the predicted Zta1 proteins were carried out using Jalview (39).Microscopic analysis of Zta1-3×GPFγ production 3×GFPγ tagged C. albicans strains were grown overnight in YPD at 30°C and then diluted 1:250 in 5 mL YPD and grown until a density of about 1 × 10 7 cells/mL.Cells (1 mL) were treated with 100 µM of one of the quinones p-benzoquinone (Sigma-Aldrich, St. Louis, MO), 2-tert-butyl-1,4-benzoquinone (Cayman Chemical, Ann Arbor, MI), menadione (MND; Sigma-Aldrich, St. Louis, MO) or 500 µM of H 2 O 2 and incubated for 1 h at 30°C on a tube roller.Samples were centrifuged, washed in sterile phosphate-buffered saline (PBS), and analyzed by fluorescence microscopy.To assess the effect on Zta1 production at different times and concentrations of incubation with BZQ, cells were treated with 10, 30, or 100 µM of BZQ, or treated with 100 µM of BZQ for 15, 30, and 60 minutes of incubation and then prepared for imaging as described above.Zeiss ZEN software was used to control the microscope and for deconvoluting images and calculating the mean fluorescence intensity (MFI) of cells.Statistical analysis of MFI compared to non-treated GFP-tagged cells was conducted with Prism 6 software (GraphPad Software, Inc., La Jolla, CA).

Western blot analysis of Zta1-3×GFPγ levels
The same protocol described above was used for western blot analysis.Briefly, 1 × 10 7 cells/mL were treated with 100 µM of BZQ, TBBQ, MEN, or 500 µM of H 2 O 2 and incubated for 1 h at 30°C with shaking before harvesting.The same process was used for cells treated with 10, 30, or 100 µM of BZQ for 1 h or cells treated with 100 µM of BZQ for 15, 30, or 60 minutes.Cell lysates were prepared by bead-bashing as follows: 20 mL of cell culture prepared as described above were centrifuged, washed with PBS, and lysed using 300 µL of 1× Laemmli buffer (2% SDS, 10% glycerol, 125 mM Tris-HCl, pH 6.8, and 0.002% bromophenol blue, 5% 2-mercaptoethanol) and zirconia beads by five rounds of 1 minute of bead beating followed by 1 minute on ice.Samples (10 µL) were separated by SDS-PAGE and transferred to a 0.4 mm nitrocellulose membrane using a semidry transfer apparatus.Blots were probed for 1 h at room temperature (RT) with a mouse anti-GFP antibody (Living Colors -JL-8, BD Biosciences Clontech) in TBS-T buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, 2% [wt/vol] bovine serum albumin [BSA], and 0.2% [wt/vol] sodium azide).The blots were then washed and incubated for 1 h with an IRDye 800-conjugated anti-mouse IgG (LI-COR Biosciences, Lincoln, NE) diluted 1:15,000 in TBS (20 mM Tris, 150 mM NaCl) containing 0.5% Tween 20.Blots were washed using TBS-T and visualized by scanning with an Odyssey CLx infrared imaging system (LI-COR Biosciences).The resulting images were analyzed using Image Studio software (LI-COR Biosciences).For Coomassie-stained gels, SDS-PAGE was performed as described above,

ROS accumulation
C. albicans (WT, zta1Δ/Δ, and zta1+) was grown overnight in 5 mL of YPD.The following day cultures were diluted to 0.100 OD into 5 mL of fresh YPD and grown for 2 h at 30°C agitating.BZQ or TBBQ (10, 30, and 100 µM/mL) was added, and cultures were incubated for an additional 1 h.After that, H 2 DCFDA (10 µM/mL) was added, and cultures were incubated for 20 minutes in the dark at 30°C with shaking.Cultures were centrifuged, washed twice with PBS, and resuspended in 100 µL of PBS.Samples were analyzed by microscopy at 492 nm in a Zeiss Axiovert 200M microscope equipped with an AxioCam HRm camera and Zeiss ZEN software for deconvolving images.

Human neutrophil killing of C. albicans
Blood was obtained from study participants with written informed consent through a protocol approved by the University of Wisconsin Internal Review Board.Neutrophils were isolated from four different donors using the MACSxpress Neutrophil Isolation and MACSxpress Erythrocyte Depletion kits (Miltenyi Biotec Inc., Auburn, CA) and suspended in RPMI 1640 (without phenol red) supplemented with 2% heat-inactivated fetal bovine serum (FBS) and supplemented with glutamine (0.3 mg/mL) as previously described (40).
For the killing assays, neutrophils (4 × 10 5 cells) and the C. albicans strains (1 × 10 6 cells) were added to wells of a culture-treated 96-well flat-bottom plate and incubated for 4 h at 37°C in 5% CO 2 .After incubation, 10 µg of DNAse I was added to each well and the plate was incubated at 37°C with 5% CO 2 for 10 minutes, after which contents of the wells (media and non-adherent cells) were moved into a 96-well round-bottom plate.Some neutrophils and C. albicans cells can adhere to the flat-bottom plate, so the contents of both the flat-bottom plate and the round-bottom plate were processed and combined back in the flat-bottom plate for analysis.The round-bottom plate was centrifuged at 1,200 × g for 2 minutes, the supernatant was discarded and 150 µL of ddH 2 O containing 1 µg/mL DNAse1 was added to each well of the round-bottom and the flat-bottom plate.Plates were incubated at 37°C for 20 minutes to lyse neutrophils.The round-bottom plate was centrifuged at 1,200 × g and the supernatant was removed, after which the contents of the original flat-bottom plate were removed and used to resuspend the contents of their respective wells in the round-bottom plate.The lysis step was repeated one additional time, after which the round-bottom plate was centrifuged at 1,200 × g, the supernatant removed, and the remaining yeast cells were resuspended in 100 µL of RPMI + 2% FBS and transferred to the original wells of the 96-well flat-bottom plate.Then, 10 µL PrestoBlue (Invitrogen, Eugene, OR) was added to each well, gently mixed, and the plate was incubated for 25 minutes at 37°C.Fluorescence 560/590 was then read on a BioTek Synergy|H1 microplate reader (Agilent, Santa Clara, CA).The percentage of viable C. albicans cells was quantified by calculating the fluorescence signal of the treated well (mutant and neutrophils) as a percentage of the control well (same mutant and no neutrophils).

Mouse infection assays
Fungal burden was tested in mice as previously described ( 24) using a protocol approved by the Stony Brook University IACUC committee.Strains were grown overnight in YPD medium, reinoculated into fresh medium, and incubated again overnight.Cells were harvested after centrifugation, washed twice with PBS, and counted using a hemocytom eter.Cells were diluted to 1.25 × 10 6 cells/mL with PBS.Female BALB/c mice (8 weeks old) were injected via the lateral tail vein with 2.5 × 10 5 cells (200 µL).After 48 h or 72 h, kidneys were excised, weighed, and then homogenized in 5 mL PBS for 30 s with a tissue homogenizer (Pro Scientific Inc.).The CFU per gram of kidney (CFU/g kidney) was determined by plating dilutions of the homogenates on YPD agar medium plates and incubating for 2 days at 30°C.Statistical analysis of the CFU data was carried out with Prism 6 software (GraphPad Software, Inc., La Jolla, CA) using one-way analysis of variance with one-way ANOVA.

FIG 1
FIG 1 Conservation of Zta1 protein sequence.ClustalW alignment of the amino acid sequences of Zta1 from Candida albicans, Saccharomyces cerevisiae, and the human homolog Cryz.Identical residues are highlighted in black and similar residues are shaded in gray.The red asterisk indicates Zta1 active site residues.A high degree of amino acid similarity was found at these sites.

FIG 2
FIG 2 Fluorescence microscopy of C. albicans cells producing Zta1.(A) C. albicans ZTA1-3×GFP cells were incubated with 100 µM of p-benzoquinone, 2-tert-butyl-1,4-benzoquinone, menadione, and 500 µM of hydrogen peroxide at 30°C for 1 h, washed and then imaged by fluorescence microscopy.(B) C. albicans ZTA1-3×GFP cells were treated with different concentra tions of p-benzoquinone (10, 30, and 100 µM) for 1 h and then analyzed.(C) cells were treated with 100 µM of p-benzoquinone and imaged after the indicated minutes of incubation.(D-F) Median fluorescence intensity of ZTA1-3×GFP cells treated with (D) the indicated compounds for 1 h, (E) different concentrations of p-benzoquinone for 1 h, and (F) 100 µM p-benzoquinone for the indicated times.The bars represent the average fold-change of three independent assays performed on different days.t-tests were performed comparing each condition to the control cells treated with dimethyl sulfoxide (DMSO).****, P ≤ 0.0001.

FIG 3
FIG 3 Zta1 is induced by quinones and oxidation.Western blots comparing Zta1-3×GFP production in C. albicans exposed to (A) the indicated oxidants for 1 h, (B) different concentrations of p-benzoquinone for 1 h, or (C) 100 µM of p-benzoquinone for the indicated times.Cells exposed to DMSO were used as a control.Western blots probed with anti-GFP antibodies are shown on the top, Coomassie Blue stained gels of the protein samples shown below were used as a loading control.The bar graphs underneath show that quantitation of the results was performed using Image Studio software, normalized to Coomassie-stained gels.The results represent the average of three independent experiments performed on different days.(D) CFU assay of Zta1-3×GFP tagged cells treated with 100 µM of BZQ, TBBQ, MEN, and 500 µM of H 2 O 2 for 1 h.Results represent averages from three independent experiments performed on different days.Multiple t-tests were performed comparing each condition to the DMSO-treated control.**, P ≤ 0.01; ***, P ≤ 0.001, ****, P ≤ 0.0001.t-tests were also performed comparing TBBQ with the other conditions, **, P ≤ 0.01.

FIG 4
FIG 4 ROS accumulation is higher in the zta1Δ/Δ mutant exposed to quinones.Wild-type, zta1Δ/Δ and zta1+ complemented strains were exposed to (A) 30 µM or (B) 100 µM p-benzoquinone, or (D) 30 µM or (E) 100 µM 2-tert-butyl-1,4-benzoquinone for 1 h and then the accumulation of ROS was assayed by incubating the cells with 2′,7′-dichlorofluorescein diacetate for 20 minutes before imaging by fluorescence microscopy.(C) Median fluorescence intensity for panels A and B. (F) Median fluorescence intensity for panels D and E. Bars represent the average median fluorescence intensity of three independent assays on different days.

FIG 5
FIG 5 Zta1 acts in combination with FLPs to promote resistance to 2-tert-butyl-1,4-benzoquinone.Quantification of disk diffusion halo assays comparing the susceptibility of C. albicans strains to (A) p-benzoquinone, (B) menadione, and (C) 2-tert-butyl-1,4-benzoquinone.The x-axis indicates the concentration of the compound applied to the disk, and the y-axis indicates the diameter of the zone of growth inhibition.The strains tested included the wild-type control strain (LLF100), zta1Δ/Δ, zta1+, Δ/Δ/Δ/Δ, Q Mut, and Q Mut+ complemented strain (see Table 1).Results represent the averages from at least three independent experiments, each done in duplicate.Error bars indicate standard deviation (SD).

FIG 6
FIG6 The zta1Δ/Δ mutant does not show increased susceptibility to peroxides.Quantification of disk diffusion halo assays comparing the susceptibility of C. albicans strains to tert-butyl-hydroperoxide and hydrogen peroxide.The strains tested included the wild-type control strain (LLF100), zta1Δ/Δ, zta1+, Δ/Δ/Δ/Δ, Q Mut, and Q Mut+ complemented strain (see Table1).Results represent the averages from at least three independent experiments, each done in duplicate.Error bars indicate SD.

FIG 7
FIG 7 Susceptibility of C. albicans strains to attack by human neutrophils.Human neutrophils from four different donors were incubated with the wild-type control strain (LLF100), zta1Δ/Δ, zta1+, FLP mutant (Δ/Δ/Δ/Δ/), Q Mut, and Q Mut+ (complemented with zta1) during 4 h.Yeast viability and percentage of survival was determined using PrestoBlue dye.Data are presented as the mean of four independent experiments, each done in triplicate.Error bars indicate SD.Strains are described in Table1.

TABLE 2
Primers used in this study Coomassie brilliant blue solution (0.1% Coomassie R-250, 40% ethanol, 10% glacial acetic acid) overnight.Gels were destained in a destaining solution (40% methanol, 10% acetic acid) and analyzed using Image Studio software (LI-COR Biosciences).Statistical analysis of densitometry was conducted with Prism 6 software (GraphPad Software, Inc., La Jolla, CA) using t tests to compare treated with non-treated GFP-tagged cells. .albicans strains were grown overnight in YPD at 30°C diluted to 2.5 × 10 5 cells and then spread onto the surface of a synthetic complete medium agar plate.Ten microliters of each compound was applied to paper filter disks (Becton, Dickinson and Company, Sparks, MD), the disks were applied to the plate surface, incubated at 30°C for 48 h, and then the diameter of the zone of growth inhibition (halo) was measured.Compounds tested included p-benzoquinone (Sigma-Aldrich, St. Louis, MO), menadione (Sigma-Aldrich, St. Louis, MO), 2-tert-butyl-1,4-benzoquinone (Cayman Chemical, Ann Arbor, MI), hydrogen peroxide (Sigma-Aldrich, St. Louis, MO), tert-butyl-hydroperoxide (tBOOH; Acros Organics) and diamide (Sigma-Aldrich, St. Louis, MO).For the CFU assays, wild-type and the 3×GFP-tagged strains were grown overnight, harvested by centrifuga tion, and resuspended in PBS.A total of 1 × 10 7 cells/mL were inoculated in liquid YPD and treated with one of the following: 100 µM of p-benzoquinone, 100 µM 2-tertbutyl-1,4-benzoquinone, 100 µM menadione, or 500 µM of hydrogen peroxide.Tubes were incubated for 1 h at 30°C on a tube roller, after which cultures were centrifugated and cells washed twice with PBS.Serial dilutions were plated on YPD plates, incubated for 48 h at 30°C, and then colony-forming units were counted. C