Calcineurin is essential for extreme thermotolerance and the maintenance of cell membrane and wall integrity in C. auris

To identify the catalytic (Cna1) and regulatory (Cnb1) subunits of calcineurin in C. auris, we queried the Candida genome database (http://www.candidagenome.org/). Orthologs were identified in the clade I C. auris wild-type strain (AR0387) as Cna1 (B9J08_004950) and Cnb1 (B9J08_001732). Sequence analysis revealed that both subunits are highly conserved across eukaryotic species (S1A and S1B Fig). Protein domain analysis using the InterPro database (https://www.ebi.ac.uk/interpro/) showed that Cna1 harbors a conserved serine/threonine-specific protein phosphatase domain, which catalyzes the dephosphorylation of phosphoserine and phosphothreonine residues. The size of Cna1 is comparable to its orthologs in other fungal species (Fig 1A). Cnb1 was found to contain four EF-hand motifs, characteristic calcium-binding domains (Fig 1A). Structural modeling using AlphaFold3 predicted a heterodimeric calcineurin complex in C. auris consisting of Cna1 and Cnb1, featuring helix-loop-helix structures (Fig 1B) [13,14].

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Fig 1. Identification of the calcineurin complex and its roles in growth, thermotolerance, and stress responses in C. auris.

(A) Domain analysis of Cna1 and Cnb1 in Candida auris (Cau), Candida albicans (Cal), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Cryptococcus neoformans (Cn), and Homo sapiens (Hs) was performed using InterPro (https://www.ebi.ac.uk/interpro/). (B) Predicted structures of C. auris Cna1 and Cnb1 were modeled using AlphaFold 3. The protein sequences were processed in the order of Cna1 and Cnb1. The color coding is as follows: Cna1 in pink, the IPR006186 domain of Cna1 in yellow, Cnb1 in blue, and the IPR02048 domain of Cnb1 in green. (C) Qualitative spot assays display the growth of the following strains under varying temperatures: wild-type (WT; B8441), cna1∆ (YSBA99), cna1∆::CNA1 (+CNA1; YSBA110), cnb1∆ (YSBA102), cnb1∆::CNB1 (+CNB1; YSBA111), and cnb1∆ cna1∆ (YSBA172). WT and mutant strains were cultured overnight in liquid YPD medium at 30°C, serially diluted 10-fold, and spotted on YPD agar plates. Plates were incubated for 1 day at 25°C, 30°C, 37°C, or 42°C. (D) Quantitative growth rates of the WT, cna1∆, and cnb1∆ strains were measured at 30°C and 37°C using a multichannel bioreactor (Biosan Laboratories, Inc., Warren, MI) by measuring the OD₆₀₀ for 30 h. (E) WT and mutant strains were spotted on YPD plates and incubated at 30°C or 45°C for 2 days. (F) Growth rates of the WT and mutant strains were monitored at 43°C. (G) WT and mutant strains were spotted on YPD medium supplemented with stress-inducing agents, including 0.1% sodium dodecyl sulfate (SDS), 0.05% Congo red (CR), 3 mg/mL calcofluor white (CFW), 250 mM CdSO₄, 2.5 μg/mL tunicamycin (TM), or 22 mM dithiothreitol (DTT). Plates were incubated for 2 days at 30°C.

https://doi.org/10.1371/journal.ppat.1013363.g001

To investigate the roles of calcineurin in C. auris, we generated cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants in the clade I AR0387 (B8441) strain background (S2A, S2B, and S2C Fig). To confirm the phenotypic features of the mutants, we also constructed complemented strains by integrating the wild-type allele into its original locus (cna1Δ::CNA1 and cnb1Δ::CNB1) (S2D and S2E Fig). Under nutrient-rich growth conditions at ambient temperatures (25°C to 30°C), the calcineurin mutants exhibited no growth defects (Fig 1C and 1D), indicating that the calcineurin pathway is not essential for the normal growth of C. auris. As the calcineurin pathway has been implicated in thermotolerance, we assessed the growth of the mutants at elevated temperatures (37°C to 42°C) [12]. The cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants showed no growth defects at high temperatures within the physiological range (37–39°C) (Fig 1C and 1D), suggesting that the calcineurin pathway is largely dispensable for thermotolerance of C. auris under these conditions. However, under severe heat stress conditions exceeding 42°C (43–45°C), cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants exhibited significant growth defects (Fig 1E and 1F), indicating the critical role of calcineurin in regulating growth at extreme temperatures.

We next investigated whether the calcineurin pathway is required for the growth of C. auris under various environmental stress conditions, including osmotic stress, oxidative stress, genotoxic stress, and cell membrane/wall-damaging stress (Figs 1G and S3). Among these stressors, the cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants demonstrated markedly enhanced sensitivity to cell membrane-destabilizing agents [sodium dodecyl sulfate (SDS)] and cell wall damaging agents [dithiothreitol (DTT), tunicamycin (TM), Congo red (CR), calcofluor white (CFW), and CdSO₄ (CDS)] (Fig 1G). SDS induces cell membrane stress by disrupting lipid bilayers [15], while CR binds specifically to polysaccharide components such as chitin and glucans in the fungal cell wall [16]. DTT and TM, as ER stressors, cause protein misfolding, leading to compromised cell wall integrity [17]. CdSO₄, a toxic heavy metal, interferes with the synthesis and structure of fungal cell wall components such as chitin and glucans, thereby disrupting cell wall integrity [18]. Notably, deletion of both CNA1 and CNB1 resulted in phenotypes identical to those observed with individual deletions of CNA1 or CNB1, suggesting that Cna1 and Cnb1 function as components of a single calcineurin complex, as expected. In summary, our findings demonstrate that the calcineurin pathway plays a crucial role in extreme thermotolerance and the maintenance of the cell membrane and wall integrity in C. auris.

Calcineurin promotes tolerance to azoles and echinocandins in C. auris

Given the role of calcineurin in maintaining cell membrane and wall integrity, we investigated its contribution to tolerance against clinically relevant antifungal drugs, which target either the fungal cell membrane (azoles and polyenes) or cell wall (echinocandins). We evaluated the susceptibility of the mutants (cna1Δ, cnb1Δ, and cnb1Δ cna1Δ) and their complemented strains to echinocandins [caspofungin (CAF), micafungin (MIF), and anidulafungin (ANF)], polyenes [amphotericin B (AMB)], and azoles [fluconazole (FLC), posaconazole (PSC), voriconazole (VRC), ketoconazole (KTC), and itraconazole (ITC)] (Fig 2A). The cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants exhibited substantially increased susceptibility to all tested echinocandins and azoles compared to the wild-type strain (Fig 2A). The minimum inhibitory concentrations (MICs) of CAF, MIF, and ANF for the wild-type strain were > 80 μg/mL, 1.25 μg/mL, and 1.25 μg/mL, respectively (Figs 2B and S4A). In contrast, the MICs for calcineurin mutants were markedly reduced: 5 μg/mL for CAF, 0.625 μg/mL for MIF, and 0.3125 μg/mL for ANF (Figs 2B and S4A). Consistent with previous reports [19], azoles exhibited fungistatic activity against wild-type C. auris and did not completely inhibit its growth (Fig 2B). However, in the absence of calcineurin (cna1Δ, cnb1Δ, and cnb1Δ cna1Δ), azoles displayed fungicidal activity (Fig 2B).

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Fig 2. Functions of calcineurin in antifungal tolerance in C. auris.

(A) Qualitative spot assays showing the stress susceptibility of the following strains: WT (B8441), cna1∆ (YSBA99), + CNA1 (YSBA110), cnb1∆ (YSBA102), + CNB1 (YSBA111), and cnb1∆ cna1∆ (YSBA172). WT and mutant strains were cultured overnight in liquid YPD medium at 30°C, serially diluted 10-fold, and spotted on YPD agar medium supplemented with stressors, including 2.5 μg/mL caspofungin (CAF), 0.15 μg/mL micafungin (MIF), 1 μg/mL anidulafungin (ANF), 3 μg/mL amphotericin B (AMB), 150 μg/mL fluconazole (FLC), 0.5 μg/mL posaconazole (PSC), 0.8 μg/mL voriconazole (VRC), 5 μg/mL ketoconazole (KTC), or 1 μg/mL itraconazole (ITC). Plates were incubated at 30°C for 3 days. (B) EUCAST MIC test results of CAF and FLC in the WT, cna1∆, cnb1∆, and cnb1∆ cna1∆ (referred to as DKO) strains. The strains were grown in YPD medium overnight, washed twice with H₂O, resuspended in H₂O, and added (100 μL) to 10 mL of liquid RPMI 1640 medium, which was subsequently loaded into 96-well plates. Drugs serially diluted 2-fold from the indicated concentrations were added to each well. To evaluate fungicidal activity, cultures were spotted onto YPD agar plates, followed by a 24 h incubation at 30°C. (C) Checkerboard assay results of CAF with FK506 in the WT strain. The assay was performed using serial two-fold dilutions of CAF, with concentrations ranging from 0 to 20 µg/mL. After growth measurement, cultures were spotted onto YPD agar plates and incubated at 30°C for 24 h to evaluate fungicidal activity. An FIC index of <0.03 was observed. (D) Checkerboard assay results of FLC with FK506 in the WT strain. The FIC index was determined to be < 0.08. (E) qRT-PCR analysis of ERG11 and ERG6 in the WT, cna1∆, and cnb1∆ strains. The strains were cultured for 24 h in the presence of FLC (200 μg/mL) at 30°C in a shaking incubator. (F) qRT-PCR analysis of FKS1 and FKS2 in the WT, cna1∆, and cnb1∆ strains. The strains were cultured overnight at 30°C shaking incubator, sub-cultured to OD₆₀₀ 0.8 in fresh YPD liquid medium, and then cultured for 24 h with CAF (5 μg/mL) at 30°C shaking incubator. Gene expression levels were normalized to ACT1, and fold changes were calculated relative to the basal expression level in WT strain. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple-comparison test (*, P < 0.05; ****, P < 0.0001; NS, not significant).

https://doi.org/10.1371/journal.ppat.1013363.g002

The observation that the calcineurin pathway promotes tolerance to azoles and echinocandins in C. auris suggests that calcineurin inhibitors, such as FK506 (tacrolimus) and cyclosporin A (CsA), may exhibit synergistic antifungal activity when combined with these drugs. To test this hypothesis, we performed checkerboard assays with FK506, CsA, azoles, and echinocandins. As anticipated, given our findings that deletion of CNA1 and CNB1 does not impair the growth of C. auris under basal conditions, FK506 and CsA alone displayed no antifungal activity at concentrations up to 16 and 24 μg/mL, respectively (Figs 2C and S4B). However, FK506 and CsA exhibited fungicidal activity only when combined with echinocandins, suggesting a synergistic effect. The drug combinations were tested using the following echinocandin concentrations: CAF (up to 20 μg/mL), MIF (1 μg/mL), and ANF (1 μg/mL). Assuming an MIC of 16 μg/mL for FK506 and 24 μg/mL for CsA, the fractional inhibitory concentration (FIC) indices for their combinations with echinocandins were as follows: FK506 combined with CAF, MIF, and ANF yielded FIC indices of <0.03, 0.14, and <0.08, respectively. Similarly, CsA combined with CAF, MIF, and ANF yielded FIC indices of <0.13, 0.14, and <0.1. All FIC indices were consistently below 0.5 (Figs 2C, S4B and S4C), indicating strong synergistic interactions. Additionally, FK506 and CsA exhibited synergistic fungicidal antifungal activity when combined with azoles. The FIC indices were <0.08 (FK506 + FLC), < 0.08 (CsA + FLC), < 0.03 (FK506 + PSC), < 0.04 (CsA + PSC), < 0.15 (FK506 + VRC), < 0.16 (CsA + VRC), 0.05 (FK506 + ITC), and 0.06 (CsA + ITC) (Figs 2D and S5).

Unexpectedly, we found that the cna1Δ, cnb1Δ, and cnb1Δ cna1Δ mutants exhibited high resistance to AMB, in stark contrast to their heightened susceptibility to azoles (Fig 2A). Azoles inhibit ergosterol synthesis by targeting lanosterol 14α-demethylase (Erg11), while AMB binds to and sequesters membrane ergosterol. A possible explanation for this finding is that the calcineurin pathway may regulate ERG11 expression in C. auris, as reduced membrane ergosterol could confer resistance to AMB. To explore this hypothesis, we assessed basal ERG11 expression levels, which were comparable between the wild-type and calcineurin mutant strains (Fig 2E). In response to FLC treatment, ERG11 expression was upregulated in the wild type, possibly as a compensatory mechanism. However, this induction was absent in the cna1Δ and cnb1Δ mutants (Fig 2E), suggesting that the calcineurin pathway plays a role in the transcriptional regulation of ergosterol biosynthetic genes associated with FLC tolerance. By contrast, ERG6 expression was induced in the calcineurin mutants upon FLC treatment, suggesting that not all ergosterol biosynthetic genes are regulated by the calcineurin pathway. In contrast, there were no appreciable changes in basal expression of ERG genes or in total ergosterol content in the calcineurin mutant strains (Figs 2E and S6). Moreover, even after AMB treatment, total ergosterol levels in calcineurin-deficient strains remained comparable to those in the wild-type strain (S6 Fig), indicating that altered ergosterol abundance is unlikely to account for the AMB resistance observed.

Interestingly, membrane permeability, which could influence azole susceptibility, was slightly increased in the calcineurin mutants, potentially promoting greater intracellular accumulation of FLC (S7 Fig). In contrast, AMB resistance may instead be attributed to changes in cell wall and membrane structure rather than ergosterol levels. These findings support the conclusion that differential responses to FLC and AMB in the calcineurin mutants are likely driven by distinct cellular mechanisms, including altered membrane permeability and cell wall architecture.

We further investigated echinocandin tolerance, which is associated with altered expression of FKS1 and FKS2, encoding β-1,3-glucan synthase, the target of echinocandins. Basal and echinocandin-induced expression levels of FKS1 in the wild type, cna1Δ, and cnb1Δ mutants, revealing no significant changes in FKS1 expression (Fig 2F). In contrast, FKS2 expression was markedly upregulated in the cna1Δ mutant upon echinocandin treatment (Fig 2F), probably as a compensatory response aimed at restoring cell wall integrity in the absence of calcineurin function (Fig 2F). The increased susceptibility of the cna1Δ mutant to echinocandins is more likely attributable to an inherent defect in maintaining cell wall integrity resulting from the loss of calcineurin function. Collectively, our findings demonstrated that the calcineurin pathway is critical for promoting tolerance to azoles and echinocandins in C. auris.

Conserved roles of calcineurin in other C. auris clades

Candida auris is classified into five clades according to the isolated location: clade I (South Asia), clade II (East Asia), clade III (Africa), clade IV (South America), and clade V (Iran) [20]. Each clade exhibits distinct genotypic and phenotypic variations [21,22]. To determine whether the role of the calcineurin pathway is conserved among different clades of C. auris, we performed a phylogenetic analysis, which revealed that Cna1 is highly conserved across all clades (Fig 3A).

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Fig 3. Functional roles of calcineurin across C. auris clades.

(A) Phylogenetic analysis of Cna1 orthologs across C. albicans, C. haemulonii, and four C. auris clades. (B) Qualitative spot assays showing the stress susceptibility of clade I WT (B8441), clade I cna1∆ (YSBA99), clade II WT (B11220), clade II cna1∆ (YSBA332), clade III WT (B11221), clade III cna1∆ (YSBA336), clade IV WT (B11245), and clade IV cna1∆ (YSBA365) strains. WT and mutant strains were cultured overnight in liquid YPD medium at 30°C, serially diluted 10-fold, and spotted on YPD medium supplemented with stressors, including 3 mg/mL CFW, 0.2% SDS, 0.05 μg/mL PSC, or 0.1 μg/mL CAF. (C) Phenome heat map of cna1∆ mutants across four different C. auris clades. Phenotype scores are color-coded based on qualitative or semi-quantitative measurements under the indicated growth conditions. Abbreviations: 30T, 30°C; 37T, 37°C; 42T, 42°C; AMB, amphotericin B; FDX, fludioxonil; 5FC, 5-flucytosine; FLC, fluconazole; PSC, posaconazole; ITC, itraconazole; VRC, voriconazole; KTC, ketoconazole; CAF, caspofungin; MIF, micafungin; ANF, anidulafungin; HPX, hydrogen peroxide; TBH, tert-butyl hydroperoxide; DIA, diamide; MD, menadione; MMS, methyl methanesulfonate; HU, hydroxyurea; TM, tunicamycin; DTT, dithiothreitol; CR, Congo red; CFW, calcofluor white; SDS, sodium dodecyl sulfate; CDS, cadmium sulfate; KCR, YPD + KCl; NCR, YPD + NaCl; SBR, YPD + sorbitol; KCS, YP + KCl; NCS, YP + NaCl; SBS, YP + sorbitol. Red and blue gradients represent phenotype reduction and enhancement, respectively, with strong, intermediate and weak phenotypes indicated by color intensity. (D) Growth in Sabouraud Dextrose (SabDex) medium showing cell aggregation in the WT and cna1∆ strain across different clades.

https://doi.org/10.1371/journal.ppat.1013363.g003

To validate the functions of Cna1 in clades other than clade I, we constructed CNA1 knockout mutants in strains B11220 (clade II), B11221 (clade III), and B11245 (clade IV) (S8 Fig). Spot assays were conducted to evaluate the mutants’ responses to various stress conditions. The stress response phenotypes of these mutants were largely consistent with those of B8441 (clade I), except for differences observed in their sensitivity to the cell wall-destabilizing agent, CFW (Fig 3B and 3C). Unlike cna1Δ mutants in clades I and II, cna1Δ mutants in clades III and IV exhibited increased resistance to CFW, implying that the calcineurin pathway may have clade-specific roles in maintaining cell wall integrity in C. auris.

To investigate whether the phenotypic variation observed among cna1∆ mutants could be attributed to differences in CNA1 expression, we measured both basal and CFW-induced CNA1 transcript levels in wild-type strains from each clade. Upon CFW treatment, CNA1 expression was moderately upregulated across all clades, with no substantial differences observed in either basal or CFW-induced levels (S9 Fig). These results suggest that the clade-specific phenotypes are unlikely to result from differential CNA1 expression, but rather may reflect variation in downstream components or modulators of the calcineurin pathway.

Cell aggregation is a clade-specific phenotypic trait in C. auris, particularly prominent in clade III, where it is recognized as a key feature influencing virulence, skin colonization, and biofilm formation [23]. To explore the role of calcineurin in this trait, we examined the aggregation ability of the cna1∆ mutants across different clades. Notably, the clade III and IV cna1∆ mutants exhibited enhanced aggregation compared to the wild-type strain, with the clade III cna1∆ mutant showing the most pronounced effect (Fig 3D). This enhanced aggregation might contribute to its increased resistance to CFW (Fig 3C). All these findings demonstrate that the calcineurin pathway plays both evolutionarily conserved and clade-specific divergent roles in C. auris.

Crz1 is the transcription factor downstream of calcineurin in C. auris

To identify transcription factors downstream of calcineurin, we queried the Candida genome database and discovered two potential Crz1 orthologs in C. auris: B9J08_002096 and B9J08_000447. B9J08_002096 was designated as Crz1 because it shares a closer phylogenetic relationship with C. albicans Crz1 than B9J08_000447, whose homolog in C. albicans is Crz2 (S10A Fig). Both Crz1 and Crz2 contain conserved zinc finger C₂H₂ domains, which are essential for DNA binding (S10B, S10C, and S10D Fig). To elucidate the roles of Crz1 and Crz2 in C. auris, we generated crz1Δ, crz2Δ, and crz1Δ crz2Δ mutants using the B8441 strain as the parental strain (S11A, S11B, and S11C Fig).

The crz1Δ mutant displayed no growth defects at temperatures up to 42°C, similar to the calcineurin mutants (Fig 4A and 4C). However, at temperatures exceeding 42°C, the crz1Δ mutant exhibited impaired growth, suggesting that Crz1, like calcineurin, contributes to extreme thermotolerance (Fig 4B and 4C). Notably, the growth defect in the crz1Δ mutant was less pronounced than that observed in the calcineurin mutants (Fig 4B and 4C). These findings suggest that while Crz1 plays a supporting role in extreme thermotolerance, calcineurin appears to have a broader and more critical function.

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Fig 4. Crz1 functions as the downstream transcription factor of calcineurin in C. auris.

(A) Qualitative spot assays display the growth of the following strains under varying temperatures: wild-type (WT; B8441), crz1∆ (YSBA105), crz1∆::CRZ1 (+CRZ1; YSBA158), crz2∆ (YSBA143), crz1∆ crz2∆ (YSBA153), and cnb1∆ cna1∆ (YSBA172). WT and mutant strains were cultured overnight in liquid YPD medium at 30°C, serially diluted 10-fold, and spotted on YPD agar plates. Plates were incubated for 1 day at 25°C, 30°C, 37°C, or 42°C. (B) WT and mutant strains were spotted on YPD plates and incubated at 30°C or 45°C for 2 days. (C) Quantitative growth rates of the WT, cna1∆ cnb1∆, and crz1∆ strains were measured at 30°C, 37°C, and 43°C using a multichannel bioreactor (Biosan Laboratories, Inc., Warren, MI) by measuring the OD₆₀₀ for 30 h. (D) Qualitative spot assays showing the stress susceptibility of WT (B8441), crz1∆ (YSBA105), crz1∆::CRZ1 (YSBA158), crz2∆ (YSBA143), crz1∆ crz2∆ (YSBA153), and cnb1∆ cna1∆ (YSBA172) strains. WT and mutant strains were grown overnight in liquid YPD medium at 30°C, serially diluted 10-fold, and spotted on YPD medium containing stressors, including 3 μg/mL FDX, 150 μg/mL FLC, 0.1% SDS, 3.6 μg/mL TM, and 22 mM DTT. Plates were incubated for 2 days at 30°C. (E) Nuclear translocation of Crz1-mCherry upon FLC treatment. Cells were cultured overnight, synchronized to an OD₆₀₀ of 0.2, and further grown until an OD₆₀₀ of 0.8 was reached. Cells were treated with 400 μg/mL FLC for 3 h, harvested (1 mL), fixed, stained with Hoechst, and imaged using a fluorescence microscope. The white scale bar represents 10 μm. (F) Bar chart quantifying the proportion of cells exhibiting Crz1-mCherry localization in either the nucleus or cytoplasm. A total of 50 cells were counted (n = 50). Statistical significance was evaluated using two-way ANOVA Analysis. Data are presented as mean ± SEM (NS, no significant; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013363.g004

The crz1Δ mutant displayed moderate sensitivity to fludioxonil and ER stress inducers, such as tunicamycin and DTT, compared to the calcineurin mutant (Fig 4D). Additionally, the crz1Δ mutant exhibited highly increased sensitivity to cell membrane stress induced by SDS, comparable to that observed in the cnb1Δ cna1Δ mutant (Fig 4D). Moreover, the crz1Δ mutant was more susceptible to azole drugs, including FLC, PSC, VRC, and KTC, than the wild-type strain, exhibiting phenotypes similar to the cnb1Δ cna1Δ mutant but with reduced severity (Figs 4D and S12A). Complementation with the wild-type CRZ1 allele restored normal phenotypes in the crz1Δ mutant (Figs 4D and S12A). In contrast, the crz2Δ mutant exhibited minor phenotypic alterations (Fig 4D). Furthermore, the crz1∆ crz2∆ double mutant exhibited phenotypes identical to those of the crz1∆ mutant alone. All these data suggest that Crz1, but not Crz2, likely functions as the downstream effector of calcineurin in C. auris.

In several other fungi, activated calcineurin dephosphorylates Crz1, facilitating its translocation into the nucleus [5]. To investigate the cellular localization of Crz1 in C. auris, we constructed the crz1∆::CRZ1-mCherry strain, in which the crz1∆ mutant was complemented with a C-terminal mCherry-tagged CRZ1 allele (S13A Fig). The CRZ1-mCherry allele was functional, as it restored wild-type phenotypes in the crz1∆ mutant (S13C Fig). Upon FLC treatment for up to 4 h, the Crz1-mCherry protein predominantly localized to the nucleus after 3 h (Fig 4E and 4F). To confirm that calcineurin mediates Crz1 nuclear translocation, we deleted CNA1 in the Crz1-mCherry tagged complemented strain (S13 Fig). In the absence of CNA1, Crz1 failed to localize to the nucleus in response to FLC (Fig 4E and 4F). These findings demonstrate that Crz1 functions as the downstream transcription factor of calcineurin and undergoes nuclear translocation in response to azoles in a calcineurin-dependent manner.

Crz1 and calcineurin play distinct roles in resistance to cell wall-damaging stressors and echinocandins in C. auris

We identified an unexpected phenotypic difference between the crz1∆ and calcineurin mutants. The crz1Δ mutant exhibited markedly increased resistance to cell wall-damaging stressor CFW and echinocandins, including CAF, MIF, and ANF, which was in stark contrast to the heightened susceptibility observed in the cna1Δ and cnb1Δ mutants (Figs 5A and S12). Interestingly, Crz1 was able to undergo nuclear translocation in response to CAF in a calcineurin-dependent manner (Fig 5B and 5C). These seemingly contradictory findings suggest that Crz1 may act as both a transcriptional activator and repressor, depending on the external cues and environmental context. Alternatively, Crz1 may regulate other transcription factors that, in turn, modulate genes involved in maintaining cell wall integrity in C. auris.

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Fig 5. Crz1 opposes calcineurin in regulating echinocandin resistance in C. auris.

(A) Spot assays showing the growth of WT and mutant strains on YPD medium containing 2.5 μg/mL CAF, 1 μg/mL ANF, or 3 mg/mL CFW. Plates were incubated for 2 days at 30°C. (B) Nuclear translocation of Crz1-mCherry upon treatment with 10 μg/mL CAF for 30 min. (C) Bar chart quantifying the proportion of cells exhibiting Crz1-mCherry localization in either the nucleus or cytoplasm. A total of 50 cells were analyzed (n = 50). Statistical significance was determined using two-way ANOVA. Data are shown as mean ± SEM (NS, no significant; ****, P < 0.0001). (D, F) Chitin and chito-oligomers staining in the WT and mutant strains. Cells were grown overnight at 30°C in liquid YPD medium, subcultured to an OD₆₀₀ of 0.8, and stained with wheat germ agglutinin (WGA) or CFW. Representative fluorescence microscopy images are displayed for each strain (scale bar, 10 μm). (E, G) Quantitative fluorescence measurements of at least 50 individual cells per strain, analyzed using ImageJ/Fiji software, are shown. (H) qRT-PCR analysis of chitin synthase genes (CHS1-CHS7) and chitin deacetylase gene (CDA2) in WT, cna1∆, cnb1∆, and crz1∆ strains. Cells were cultured for 24 h with 5 μg/mL CAF at 30°C in a shaking incubator. Gene expression levels were normalized to ACT1, and fold changes were calculated relative to the basal expression level in WT. (E, G, H) Statistical significance was evaluated using one-way ANOVA with Bonferroni’s multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant).

https://doi.org/10.1371/journal.ppat.1013363.g005

These findings suggest that calcineurin and Crz1 may play distinct roles in maintaining fungal cell wall integrity, as CFW and echinocandins disrupt chitin and β-glucan layers, respectively, which are major components of the fungal cell wall [24]. To further investigate the differential roles of calcineurin and Crz1 in maintaining cell wall integrity, we analyzed CFW and wheat germ agglutinin (WGA) staining patterns in the wild-type, calcineurin (cnb1∆ cna1∆), and crz1∆ mutant strains. CFW binds to β-linked polysaccharides, such as chitin and chitosan, while WGA with high molecular weight specifically binds to surface-exposed chito-oligomers [25,26]. CFW staining revealed increased fluorescence in both calcineurin and crz1Δ mutants compared to the wild type, indicating enhanced chitin accumulation in these mutants (Fig 5D and 5E). However, WGA staining showed that fluorescence levels in the calcineurin mutants were similar to those of the wild type, whereas the crz1Δ mutant exhibited significantly reduced fluorescence (Fig 5F and 5G). These results suggest that deletion of CRZ1, but not calcineurin, reduces chito-oligomer exposure.

To further investigate the roles of calcineurin and Crz1 in chitin biosynthesis, we measured the expression levels of the chitin synthase (CHS) and chitin deacetylase (CDA) genes. Under basal conditions, no significant changes in expression levels were observed (Fig 5H). However, upon treatment with CAF, the expression levels of chitin synthase genes (CHS1-CHS7) and CDA2 were significantly upregulated in the cna1∆ or cnb1∆ mutants compared to the wild-type strain, whereas the crz1∆ mutant showed no notable induction (Fig 5H). These findings suggest that calcineurin mutants compensate for this heightened echinocandin susceptibility by upregulating chitin synthesis genes to reinforce the cell wall. In contrast, the crz1∆ mutant, which exhibits resistance to echinocandins, did not induce chitin synthesis under these conditions (Fig 5H). These findings underscore the complexity of the calcineurin/Crz1 pathway in maintaining fungal cell wall integrity.

To assess whether the induction of CHS genes in the cna1Δ mutant upon CAF exposure is mediated by the PKC-dependent cell wall integrity pathway, we generated a cna1Δ mkc1Δ double mutant by deleting MKC1 (B9J08_002682), a mitogen-activated protein kinase (MAPK) central to the PKC pathway in C. albicans [27], in the cna1Δ background. Quantitative gene expression analysis revealed that CHS gene upregulation was further enhanced in the cna1Δ mkc1Δ mutant compared to the cna1Δ single mutant following CAF treatment (S14 Fig). These results demonstrate that the increased CHS expression in the absence of calcineurin is not positively regulated by the PKC-Mkc1 MAPK cascade. Instead, they implicate the involvement of additional PKC-independent signaling pathways that are activated in response to cell wall stress when calcineurin function is impaired.

The calcineurin pathway is dispensable for morphogenesis, biofilm formation, secreted aspartyl protease activity, and ploidy switching in C. auris

We next examined whether the calcineurin/Crz1 pathway influences other known virulence traits in C. auris. Morphological changes, critical virulence factors in Candida species, were assessed by inducing pseudohyphal formation with the DNA-damaging agent hydroxyurea [28]. Both calcineurin and crz1Δ mutants exhibited no significant differences in pseudohyphal formation compared to the wild type (S15A Fig). Biofilm formation and the production of secreted aspartyl proteases (SAPs) were also analyzed in these mutants. Our findings confirmed that the calcineurin/Crz1 pathway does not play a role in either process (S15B and S15C Fig).

In addition, we investigated the frequency of ploidy switching, another virulence trait in C. auris [29]. It has been reported that haploid and diploid C. auris cells grown on YPD medium containing phloxine B display white and pink colony phenotypes, respectively [29]. Interestingly, most calcineurin mutants, but not the crz1∆ mutant, formed pink colonies (S15D Fig). However, fluorescence-activated cell sorting (FACS) analysis revealed that the pink-colored calcineurin mutant cells were not diploid (S15D Fig), suggesting that ploidy switching did not occur in these strains. We hypothesized that the pink colony phenotype may result from altered cell membrane or wall composition, which allows the dye to penetrate more easily in the calcineurin mutant strains compared to the wild-type strain. Collectively, these results demonstrate that the calcineurin/Crz1 pathway is not involved in morphogenesis, biofilm formation, secreted aspartyl protease activity, and ploidy switching in C. auris.

Roles of the calcineurin pathway in the pathogenicity of C. auris

To determine the role of the calcineurin pathway in the pathogenicity of C. auris, we first utilized the Drosophila melanogaster infection model, a well-established fungal infection system, recognized for its short lifecycle, cost-effectiveness, ease of handling, and genetic consistency [30]. A total of 80 flies per group were injected with wild-type, cna1∆, cna1∆::CNA1, or bcy1∆ strains, and the survival rates were monitored over four days (Fig 6A). The bcy1∆ (YSBA4) strain, which lacks the regulatory subunit of protein kinase A (PKA) in the cAMP pathway, was included as a control strain due to its previously reported attenuated virulence in murine systemic infection models [31]. Flies infected with the cna1∆ mutants exhibited a significantly higher survival rate than those infected with the wild-type and cna1∆::CNA1 strains. However, this increased survival was less pronounced than in flies infected with the bcy1∆ control strain (Fig 6B). These results indicate that the calcineurin pathway is required for the virulence of C. auris in the insect infection model.

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Fig 6. The role of the calcineurin pathway in the pathogenicity of C. auris.

(A) Schematic representation of the experiment processes for the Drosophila melanogaster infection model. This figure was created using BioRender (https://biorender.com/). (B) Survival rates of D. melanogaster infected with WT, cna1∆, + CNA1, or bcy1∆ strains were monitored over 4 days post-infection (dpi). A total of 80 files were used per strain. Survival curves were statistically analyzed using the log-rank (Mantel-Cox) test. The PBS control is represented in brown. (C) Survival rates of D. melanogaster infected with WT, crz1∆, + CRZ1, or bcy1∆ strains. (D) Schematic diagram depicting an ex vivo mouse ear infection model. This figure was generated using BioRender (https://biorender.com/). (E) Equal-sized ex vivo mouse ear tissues (n = 6) were incubated with PBS-diluted C. auris cells on a rotator to allow adhesion. After incubation, adherent cells were gently scraped off, and genomic DNA was extracted. Quantitative PCR targeting the CNB1 gene was performed, and Ct values were normalized to those of the non-infected PBS-treated group. (F) After 1-h incubation, ear tissues were placed on YPD agar plates and incubated at room temperature for 2 days, followed by imaging. Scale bar indicates 0.5 mm. (G) Schematic diagram of the mouse skin infection model. This figure was designed using BioRender (https://biorender.com/). (H) A total of 10⁷ cells from WT or mutant strains were subcutaneously injected into the left and right flanks of 7-week-old BALB/c mice (n = 8). (I) Clinical scores were determined by measuring abscess areas. Statistical significance was calculated using an unpaired t-test (*, P = 0.0424). (J) Fungal burden was assessed by homogenizing abscess tissue 9 dpi and plating the homogenate on YPD agar plate. Relative fungal burden values were calculated as mutant CFU/g per WT CFU/g. Statistical significance was calculated using an unpaired t-test (***, P < 0.0005). (K, L) Survival curve and body weight changes of 7-week-old BALB/c mice (n = 7) intravenously injected with 10⁷ C. auris cells. (M) Fungal burden in the inner ear was measured 7 days post-intravenous systemic infection. Mice were treated with FLC (20 mg/kg) and CAF (2 mg/kg) via intraperitoneal injection, administered once daily for six days, starting on the day of infection. Statistical significance was calculated using an unpaired t-test (**, P < 0.01; ***, P < 0.001).

https://doi.org/10.1371/journal.ppat.1013363.g006

To determine whether the attenuated virulence observed in the cna1∆ mutant is mediated through Crz1, a downstream transcription factor of calcineurin, we assessed the virulence of the crz1∆ mutant using the same Drosophila infection model. Notably, the survival rates of flies infected with the crz1∆ mutant were comparable to those infected with the wild-type strain, indicating that loss of CRZ1 does not affect virulence in this model (Fig 6C). These findings suggest that the calcineurin-dependent regulation of C. auris virulence is largely Crz1-independent and may involve alternative downstream targets or pathways.

Next, an ex vivo skin adhesion model was utilized to evaluate the ability of C. auris to attach to mammalian skin tissue, a critical step in its colonization and infection process. Adhesion of each strain to murine ear skin tissue was quantified, and the cna1Δ mutant showed significantly reduced adhesion at the early 1-h time point compared to the wild-type and complemented strains. However, no significant difference was observed after 2 h of incubation (Fig 6E). These results suggest that calcineurin signaling is particularly important during the early stage of C. auris tissue attachment.

We next evaluated the subcutaneous and systemic infectivity of the calcineurin mutants using a murine (BALB/c) infection model. The fungal burdens in infected skin regions were quantified 9 days post-infection (dpi). Mice infected with the cna1∆ mutant exhibited significantly reduced abscess size and fungal burden compared to those infected with the wild-type strain (Fig 6H, 6I and 6J). In contrast, mice infected with the crz1∆ mutant showed no significant differences in fungal burden relative to the wild type (Fig 6J). These results highlight the critical role of Cna1 as a virulence factor necessary for C. auris skin colonization, whereas Crz1 appears dispensable in this context.

Notably, the calcineurin mutant displayed a distinct virulence pattern in a systemic murine infection model compared to the skin infection model. Mice intravenously infected with the cna1∆ mutant exhibited survival rates similar to those infected with the wild-type or cna1∆::CNA1 strain (Fig 6K). However, noticeable body weight loss in cna1∆-infected mice became apparent after 8 dpi, in contrast to mice infected with the wild-type or cna1∆::CNA1 strains (Fig 6L). In a systemic infection model treated with FLC and CAF, the fungal burden of the cna1∆ mutant in the inner ear was significantly more reduced by FLC compared to the wild-type strain (Fig 6M). Collectively, these findings suggest that the calcineurin pathway plays distinct roles in the virulence of C. auris, depending on the specific host niche.

Ethics statement

All animal care and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Experimental Animal Center at Jeonbuk National University (Approval number JBNU 2023–131). All experiments were conducted in full compliance with established ethical guidelines for animal research.

Candida auris strains and growth media

The Candida auris strains used in this research are listed in S1 Table in the supporting information. The parental wild-type strains, including B8441 (clade I, AR0387), B11220 (clade II, AR0381), B11221 (clade III, AR0383), and B11245 (clade IV, AR0386), were provided by the Centers for Disease Control and Prevention (CDC). Both wild-type and mutant strains were maintained as frozen stocks in 20% glycerol at −80°C until use. Yeast strains were cultured on YPD agar plates (2% agar in YPD broth: 2% peptone, 1% yeast extract, and 2% D-glucose) at 30°C for 24–48 h. For liquid culture preparation, yeast cells were grown in YPD media at 30°C with constant agitation at 200 rpm. Before experimental assays, cells were transferred to fresh YPD broth and cultured to the mid-log phase (OD₆₀₀ = 0.6–0.8) prior to the designated treatments.

Gene deletion and complementation

Gene deletion mutants were constructed using the nourseothricin resistance (CaNAT), hygromycin B resistance (CaHYG), or G418 resistance (CaNEO) markers, each flanked by 0.5–0.7 kb 5′ and 3′ regions of the target genes, including CNA1, CNB1, CRZ1, and CRZ2. Gene disruption cassettes containing the selection markers were assembled through double-joint PCR. In the first round of PCR, the flanking regions of each target gene were amplified using the L1-L2 and R1-R2 primer pairs. The CaNAT selection marker was amplified from the plasmid pV1025, which contains the CaNAT gene, using the appropriate primer pairs listed in S1 Table in the supporting information. Similarly, the CaNEO selection marker was amplified from the plasmid pTO149 RFP-NEO, which carries the CaNEO gene, using corresponding primer pairs. The PCR products of the flanking regions and selection markers were purified and subsequently used as templates for a second round of double-joint PCR. During this step, the 5′- and 3′-gene disruption cassettes containing split selection markers were amplified using the L1-split primer 2 and R2-split primer 1, respectively (see S1 Table in the supporting information).

Gene disruption cassettes were introduced into C. auris using a modified lithium acetate/heat-shock transformation protocol. Overnight cultures were grown at 30°C in 50 mL of YPD broth with constant shaking. A 1.2 mL aliquot of cultured cells was harvested by centrifugation, washed sequentially with dH₂O and lithium acetate buffer (100 mM lithium acetate, 10 mM Tris, 1 mM EDTA, pH 7.5), and resuspended in 300 μL of lithium acetate buffer. For each transformation, the reaction mixture consisted of 10 μL of denatured salmon sperm DNA (Sigma, cat no. D9156), 100 μL of the cells, 500 μL of 50% PEG4000 (Sigma, cat no. P4338), and 50 μL of the amplified gene deletion cassette. The mixture was incubated at 30°C for 6 h with intermittent inverting, followed by a 20-min heat shock at 42°C and immediate cooling on ice for 1 min. The cells were then pelleted, resuspended in 1 mL of YPD medium, and incubated at 30°C for 1–2 h with shaking to allow recovery. After recovery, the cells were washed twice with fresh liquid YPD medium and plated onto selective YPD agar containing 400 μg/mL nourseothricin, 1.8 mg/mL hygromycin B, or 2.4 mg/mL G418. Plates were incubated at 37°C for 2–3 days to select transformants. Positive transformants resistant to nourseothricin, hygromycin B, or G418 were verified for the correct genotype using diagnostic PCR and confirmed via Southern blot analysis.

To validate the phenotypes of the cna1Δ, cnb1Δ, and crz1Δ mutants, we generated corresponding complemented strains by re-integrating the respective wild-type genes into their native loci (cna1Δ::CNA1, cnb1Δ::CNB1, and crz1Δ::CRZ1). Full-length gene fragments were amplified by Pfu-PCR using genomic DNA extracted from the wild-type B8441 strain as a template, along with the primer pairs listed in S2 Table in the supporting information. The amplified fragments were cloned into the TOPO vector (Invitrogen), resulting in the plasmids pTOP-CNA1, pTOP-CNB1, and pTOP-CRZ1. After sequence verification, each plasmid was modified by subcloning the CaHYG selection marker into the corresponding pTOP vector, generating pTOP-CNA1-HYG, pTOP-CNB1-HYG, and pTOP-CRZ1-HYG. For re-integration into the native locus, pTOP-CNA1-HYG, pTOP-CNB1-HYG, and pTOP-CRZ1-HYG were linearized using PmlI, BsmI, and BglII, respectively, and introduced into the corresponding mutant strains using the lithium acetate heat-shock transformation protocol. The successful generation of complemented strains was confirmed through diagnostic PCR, verifying the correct integration of the wild-type alleles.

Total RNA preparation and quantitative RT-PCR

Total RNA was extracted from C. auris wild-type and calcineurin/Crz1 mutant strains cultured overnight at 30°C in YPD broth. Cells were harvested by centrifugation at mid-log phase (OD₆₀₀ of 0.6–0.8), rapidly frozen in liquid nitrogen, and lyophilized. For RNA isolation, 30 mL of the culture was maintained as the basal condition, while the remaining 30 mL was subjected to additional incubation with specific stress agents. Total RNA was extracted using the Trizol-based Easy Blue reagent (Intron). Complementary DNA (cDNA) was synthesized from purified total RNA using reverse transcriptase (Thermo Scientific) following the manufacturer’s protocol. Quantitative PCR (qPCR) was performed using a CFX96 Real-Time system (Bio-Rad) with gene-specific primer pairs. Expression levels were normalized to the housekeeping gene ACT1. Statistical analyses were conducted using one-way ANOVA followed by Bonferroni’s multiple-comparison test. To ensure reproducibility, all experiments were performed in technical triplicates and independently repeated three times with biological replicates.

Growth and stress sensitivity spot assay

To evaluate the growth and stress sensitivity of wild-type and calcineurin/Crz1 mutant strains of C. auris, cells were cultured overnight at 30°C, serially diluted 10-fold up to four times (final dilution 1:10⁴), and spotted onto YPD plates. The plates were incubated at various temperatures (25°C, 30°C, 37°C, 42°C, and 45°C), and growth was qualitatively assessed by photographing the plates after 24 h of incubation. To assess stress responses, specific chemical stressors were incorporated into the growth media, including an osmotic stressor (sorbitol), cation and salt stressors (NaCl or KCl), oxidative stressors [hydrogen peroxide (HPX), tert-butyl hydroperoxide (TBH), menadione (MD), or diamide (DIA)], a membrane destabilizing stressor (SDS), cell-wall destabilizing stressors [Congo red (CR) and calcofluor white (CFW)], and various antifungal agents [fludioxonil (FDX), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), posaconazole (PSC), ketoconazole (KTC), itraconazole (ITC), caspofungin (CAF), micafungin (MIF), anidulafungin (ANF) or amphotericin B (AMB)]. The plates were grown at 30°C and photographed after 2–3 days of incubation under stress conditions.

EUCAST MIC test and checkerboard assay

Wild-type and mutant C. auris strains were cultured overnight at 30°C in YPD medium, washed twice with dH₂O, and resuspended in dH₂O. For the EUCAST (European Committee on Antimicrobial Susceptibility Testing) MIC assay, the cell suspension was adjusted to an OD₆₀₀ of 1.0. A total of 100 μL of the prepared cell suspension was added to 10 mL of 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered RPMI 1640 media (pH 7.4, supplemented with 0.165 M MOPS and 2% glucose), and the mixture was dispensed into 96-well plates containing 2-fold serial dilution of the test drugs. For the checkerboard assay, the cell suspension in RPMI medium was dispensed into 96-well plates containing 2-fold serial concentrations of each drug combination. The plates were incubated at 35°C for 48 h. Cell density in each well was measured at OD₅₉₅ to determine the MIC values. Following growth assessment, cultures from each well were spotted onto YPD agar plates and incubated at 30°C for 24 h to evaluate their fungicidal effects.

Quantification of ergosterol content using HETENE method

Ergosterol quantification was performed using a modified HETENE method. Strains were pre-cultured overnight in 50 mL of YPD medium at 30°C with shaking at 220 rpm. The overnight cultures were diluted to an OD₆₀₀ of 0.2 in fresh 50 mL YPD and sub-cultured for 3–4 h under the early exponential phage until reaching an OD₆₀₀ of 0.8–1.0. Cells were then treated with AMB (3 μg/mL) for 3 h, harvested and washed with sterile distilled water. Each pellet was resuspended in 3 mL of 25% alcoholic potassium hydroxide (prepared by dissolving 25 g of KOH in 35 mL of sterile distilled water and adjusting the volume to 100 mL with absolute ethanol) and vortexed for 1 min. The suspensions were transferred to sterile borosilicate glass screw-cap tubes (16 × 100 mm) and incubated in an 85°C water bath for 1 h, followed by cooling to room temperature. Sterols were extracted by adding 1 mL of sterile distilled water and 3 mL of n-heptane, followed by vigorous vortexing for 3 min. For quantification, 200 μL of the n-heptane layer was mixed with 800 μL of 100% ethanol (5-fold dilution). Absorbance was measured at both OD₂₈₂ and OD₂₃₀ using a UV spectrophotometer. Ergosterol content was calculated by the following equations: % ergosterol = [(OD₂₈₂/290) × F]/ dry pellet weight – [(OD₂₃₀/518) × F]/ dry pellet weight. F is the ethanol dilution factor [5]. To determine the sterol composition profile, the n-heptane (upper) layer was transferred to a clean tube and stored at –20°C for up to 24 h. For spectrophotometric analysis, 2 mL of the sterol extract was brought to room temperature to dissolve residual sterols, and absorbance was scanned from 200 to 400 nm at 1 nm intervals using a spectrophotometer.

Membrane permeabilization assay using flow cytometry

Membrane integrity was assessed by flow cytometry using propidium iodide (PI) staining. Strains were pre-cultured overnight in 50 mL of YPD medium at 30°C with shaking at 220 rpm. A subculture was initiated at an OD₆₀₀ of 0.2 in 60 mL of fresh YPD and incubated at 30°C with shaking at 220 rpm for approximately 3 h until the OD₆₀₀ reached 0.8. One milliliter of culture was collected from each sample and centrifuged at 3000 rpm for 3 min. Cells were washed twice with PBS and fixed in 70% ethanol (300 μL of cell suspension mixed with 700 μL of 100% ethanol) at 4°C overnight with gentle rotation to prevent clumping. Following fixation, cells were washed twice with PBS and treated with 100 μL of RNase A buffer (0.2 M Tris-HCl, pH 7.5; 20 mM EDTA) containing 1 μL of RNase A (from 10 mg/mL stock), then incubated at 37°C for 4 h. Cells were washed twice with PBS, cells were adjusted to a concentration of 1 × 10⁶ cells/mL in PBS. PI was added to a final concentration of 50 μg/mL, and the suspension was incubated in the dark at 25°C for exactly 15 min for all strains to ensure consistent staining conditions. Finally, the cells were washed once with PBS and resuspended in 500 μL PBS for flow cytometric analysis (CytoFLEX, Beckman Coulter).

Evaluating cell aggregation

Wild-type and mutant strains were cultured in Sabouraud Dextrose (SabDex) medium for two days, washed twice with phosphate-buffered saline (PBS), and counted. A total of 2.5 × 10⁸ cells were suspended in 5 mL PBS in sterile medical tubes, vortexed thoroughly, and photographed 5 min after vortexing [46]. Images were captured using fluorescence microscopy.

Observing the intracellular localization of mCherry-tagged proteins

Crz1-mCherry-tagged strains were grown overnight in YPD broth at 30°C. The overnight cultures were subcultured in YPD medium until an OD₆₀₀ of 0.8 was reached. The cells were treated with antifungal drugs and incubated under the designated conditions. For cell fixation, the samples were treated with a 4% paraformaldehyde solution containing 3.4% sucrose for 15 min at room temperature. The fixed cells were thoroughly washed with a buffer containing 0.1 M KPO₄ and 1.2 M sorbitol. The nucleus was visualized by staining the cells with 10 μg/mL Hoechst 33342 (Thermo Fisher, USA) for 30 min in the dark. Finally, the stained cells were examined using differential interference contrast (DIC) and fluorescence microscopy (Nikon Eclipse, Japan) to determine the subcellular localization of Crz1-mCherry fusion proteins.

Measurement of chitin and chito-oligomers content in cell walls

To measure the chitin and chito-oligomers content in the cell walls of wild-type and calcineurin/Crz1 mutant strains of C. auris, cells were cultured overnight at 30°C in a shaking incubator. The cells were harvested by centrifugation, washed, and resuspended in PBS (pH 7.5). For staining, the cells were treated with 100 μg/mL fluorescein isothiocyanate (FITC)-conjugated WGA or 25 μg/mL CFW for 30 min in the dark. Following staining, cells were washed three times with PBS and visualized by fluorescence microscopy (Olympus BX51). Fluorescence intensity from at least 50 individual cells per sample was quantified using ImageJ/Fiji software to determine relative chitin and chito-oligomers levels.

Assessment of morphological transition from yeasts to pseudohyphae

To induce pseudohyphal formation in wild-type and calcineurin/Crz1 mutant strains, cells were cultured for 24 h in YPD broth supplemented with 100 mM HU. Following pseudohyphal induction, the cells were fixed using 10% formalin and stained with 10 μg/mL Hoechst 33342 (Thermo Fisher) to visualize cellular morphology. The fixed and stained samples were incubated in the dark for 30 min, after which images were captured using fluorescence microscopy.

Crystal violet assay for biofilm formation

C. auris wild-type and mutant strains were cultured overnight at 30°C in 2 mL of YPD liquid medium. The cells were washed twice with sterile H₂O and resuspended in MOPS-buffered RPMI-1640 media (pH 7.4, 0.165 M MOPS and 2% glucose). The cell suspension was adjusted to an OD₆₀₀ of 0.5, and 200 μL of the suspension was dispensed into each well of a 96-well plate. The cultures were incubated at 37°C with shaking at 220 rpm for 24 h. After incubation, the cell suspensions were carefully removed, and the plates were dried in a dry oven at 65°C. Each well was then treated with 150 μL of 0.5% crystal violet solution and incubated at room temperature for 10 min for staining. Excess dye was removed by washing the wells three times with PBS, and the plates were dried again in the dry oven at 65°C. To solubilize the bound crystal violet, 200 μL of 33% acetic acid was added to each well and incubated for 2 min. The solubilized solution was diluted 1:10 with PBS and transferred to a new 96-well plate. Absorbance was measured at OD₅₉₅ using a microplate reader.

Secreted aspartyl proteinase activity assay

SAP activity was assessed using the yeast carbon base-bovine serum albumin (YCB-BSA) method. C. auris strains were grown overnight in 2 mL of YPD broth at 30°C, harvested by centrifugation, washed with dH₂O, and resuspended in 1 mL of dH₂O. The cell suspension was adjusted to a final concentration of 10⁵ cells/mL. Subsequently, 3 μL of the cell suspension was spotted onto YCB-BSA plates (containing 23.4 g/L yeast carbon base and 0.2% BSA) and incubated at 37°C for 3 days. SAP activity was quantified by measuring the diameter of the halo formed around the colonies. All experiments were performed in triplicate with independent biological repeats to ensure reproducibility.

Ploidy switching and flow cytometry analyses

C. auris wild-type and calcineurin/Crz1 mutant strains were cultured overnight at 30°C in YPD medium, washed twice with PBS, and counted. For the ploidy switching assay, 100 cells were spread onto YPD media supplemented with 5 μg/mL phloxine B and incubated at 25°C for 14 days. For DNA content analysis, single colonies from YPD medium containing phloxine B were inoculated into liquid YPD medium and cultured overnight at 30°C. The cells were then harvested, washed with PBS, and counted. A total of 10⁶ cells were fixed in 70% ethanol for 14–16 h at 4°C. Following fixation, cells were washed twice with PBS and treated with RNase A (200 μg/mL) at 37°C for 1 h to remove RNA contamination. After centrifugation, the supernatant was discarded, and the cells were stained with 500 μL of 100-μg/mL propidium iodide for 30 min at room temperature in the dark. The stained cells were washed with PBS, resuspended in 300 μL of PBS, and analyzed for DNA content using flow cytometry. A minimum of 10,000 cells were evaluated per experiment to ensure statistical reliability.

Drosophila melanogaster infection model

The Drosophila melanogaster (w1118) was used for infection experiments. Flies were maintained at 25°C with a 12-h light-dark cycle on standard cornmeal-agar medium obtained from Bloomington Stock Center. Cells from C. auris wild-type and mutant strains were cultured overnight at 30°C in YPD medium, followed by three washes with PBS. The cell concentration was adjusted to 3 × 10⁹ cells/mL. The cell suspensions were centrifugated, and the resulting pellets were resuspended in PBS containing 1% (w/v) blue food dye (FD&C Blue #1) to ensure visualization. For the survival assay, adult female flies (4–5 days old) were anesthetized using CO₂ and the dorsal thorax was pricked with a thin needle dipped in the prepared cell suspension. Approximately 80 flies per strain were infected, divided into groups of 10, and placed into individual vials containing standard cornmeal-agar medium. The flies were incubated at 30°C, and survival was monitored daily for up to 4 days post-infection (dpi).

Ex vivo murine ear tissue adhesion assay

An ex vivo murine ear tissue adhesion assay was performed to evaluate the adhesion capacity of C. auris strains. Mouse ears of equal size were trimmed, cleaned with PBS. For the adhesion assay, each C. auris strain was cultured overnight in 2 mL of YPD medium at 30°C with shaking at 220 rpm. Cells were harvested, washed twice with PBS, and resuspended in 10 mL of PBS and OD₆₀₀ of 0.2. Each ear was incubated in the suspension at room temperature with shaking 80 rpm for 1 or 2 h. After incubation, ears were washed three times with PBS to remove non-adherent cells, and then either incubated further on YPD plates for imaging or subject to DNA extraction for quantitative analysis. The ears were incubated in phenol:chloroform for 30 min to detach all surface-adherent fungal cells along with epidermal cells. The resulting suspension was then subjected to bead beating to extract total DNA. For quantitative analysis of adhesion, a CNB1 gene-specific primer set was used to detect WT and mutant strains by qPCR. Fungal burden was quantified by comparing Ct values with those obtained from uninfected ear tissue controls.

Subcutaneous murine infection model

C. auris wild-type and mutant strains were cultured overnight in YPD medium at 30°C with shaking. The cell concentration was adjusted to 10⁸ cells/mL, and 100 μL of the suspension in PBS was injected subcutaneously into anesthetized mice. SPF/VAF-confirmed, inbred 6-week-old female BALB/cAnNCrlOri mice (ORIENT BIO INC., South Korea) were housed for one week prior to infection. Anesthesia was induced by inhalation of isoflurane vapor (Hana Pharm. Co., Ltd., South Korea) at a flow rate of 80 cc/min. Subcutaneous injection was performed on both flanks of the mice, where the fur had been trimmed with clippers. The size of abscesses formed post-infection was quantified using ImageJ with a ruler as a reference. Colony-forming units (CFUs) were determined by surgically extracting the abscess, homogenizing the tissues, and plating the homogenates on YPD medium for colony enumeration.

Systemic murine infection model

The cell concentration was adjusted to 10⁸ cells/mL, and 100 μL of the suspension in PBS was injected intravenously into restrained mice. Humane endpoints were defined by the onset of systemic infection symptoms, including rapid weight loss, abnormal head tilt, and circling behavior. Survival was monitored and represented as a survival curve. For drug treatment groups, drugs were administered intraperitoneally starting from the day of infection. Each drug was prepared in a 5% Kolliphor solution (polyoxyl-35 castor oil:ethanol = 1:1), and the injection volume was limited to 100 μL. The stock concentration for a 20 mg/kg (mpk) dose was 4 mg/mL, while for a 2 mpk dose, the stock concentration was 0.4 mg/mL.