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Candida auris vacuolar calcium pump mediates fluconazole efflux and resistance evolution

Nature Microbiology (2026)Cite this article

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Abstract

Candida auris is an emerging fungal pathogen notable for its intrinsically high resistance to fluconazole, the most prescribed antifungal drug. However, the genetic regulators underlying fluconazole susceptibility in C. auris remain unclear. Here we performed a pooled screen of piggyBac (PB) transposition mutants and identified significant enrichment of mitochondrial genes whose inactivation reduces fluconazole susceptibility. A genome-wide genetic interaction analysis of a mitochondrial gene deletion mutant, pet309Δ, suggests that the vacuolar calcium pump homologue CDT1 (Calcium and Drug Transporter 1) is responsible for its reduced fluconazole susceptibility. Fluconazole induces significant upregulation of CDT1 through the calcineurin signalling pathway. Cdt1, beyond its canonical calcium-pumping function, has evolved another function in mediating fluconazole efflux through its fluconazole-induced, calcineurin- and ATP hydrolysis-dependent plasma membrane localization. In addition, Cdt1 accelerates the evolution of fluconazole resistance or tolerance, and its transcript levels are substantially elevated across resistant clinical isolates. Our findings reveal a neofunctionalized role for Cdt1 in mediating fluconazole efflux in C. auris.

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Fig. 1: A genetic screen identifies that inactivation of PET309 reduces fluconazole susceptibility in C. auris.
Fig. 2: Functional interactions of PET309 with CDR1 and 000175.
Fig. 3: Calcineurin-mediated plasma membrane localization of Cdt1 is essential for its calcium pump-independent function in fluconazole tolerance in C. auris.
Fig. 4: ATP hydrolysis-coupled phosphorylation of D429 is required for Cdt1’s calcium pump-independent function.
Fig. 5: Importance of CDT1 in the rapid evolution of fluconazole resistance in vitro and in fluconazole-resistant clinical isolates.

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Data availability

All data supporting the findings of this study are available within the paper and its supplementary files. The PB library sequencing data are available under BioProject accession no. PRJNA1273510. The RNA-seq data are deposited under Gene Expression Omnibus accession no. GSE300783. The whole-genome sequence data are available under BioProject accession no. PRJNA1278929. Source data are provided with this paper.

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Acknowledgements

We thank G. Huang and C. Liu for contributing clinical C. auris isolates to this study; the past and present members of Y.W.’s lab and J.G.’s lab for valuable discussions. This work was supported by the National Key Research and Development Program of China (2025YFA1310100), the National Natural Science Foundation (32370961), the Chinese Academy of Sciences Project for Young Scientists in Basic Research (YSBR-111), the Fundamental Research Funds for the Central Universities (22120250158 and 22120250374), the Open Research Fund of Basic Medicine College (JCKFKT-ZD-004), the National Medical Research Council of Singapore (OFIRG23Jul-0077), and the TRIDENT Program of Singapore (TP_23P2).

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Author notes

  1. These authors contributed equally: Yabing Song, Jinxin Chen, Junhua Wan, Jiamo Zhang.

Authors and Affiliations

  1. State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Yabing Song, Jinxin Chen, Junhua Wan, Qizheng Liu, Hanlin Zhang, Yuru Guo & Jiaxin Gao

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Jinxin Chen, Fei Sun & Jianbin Wang

  3. State Key Laboratory of Cardiovascular Diseases and Medical Innovation Center, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Jiamo Zhang, Yali Chen, Ziyu Yan & Kun Chen

  4. Shanghai Key Laboratory of Signaling and Disease Research, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Jiamo Zhang, Yali Chen, Ziyu Yan & Kun Chen

  5. A*STAR Infectious Diseases Labs (A*STAR ID Labs), Agency for Science and Technology Research (A*STAR), Singapore, Singapore

    Eve W. L. Chow & Yue Wang

  6. National Key Laboratory of Immunity and Inflammation, Institute of Immunology, Naval Medical University, Shanghai, China

    Kun Chen

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Contributions

Y.S. and J.C. performed most experiments, conducted bioinformatics analyses and interpreted data. J.W. performed the genetic interaction screens. J.Z. performed the experimental evolution assays. F.S. performed LC–MS experiments and interpreted data. All other authors aided in constructing plasmids and strains used in this study. K.C., Y.W. and J.G. conceptualized the study, designed the experiments and wrote the manuscript. All authors provided comments in editing the manuscript.

Corresponding authors

Correspondence to Kun Chen, Yue Wang or Jiaxin Gao.

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Nature Microbiology thanks Yong-Sun Bahn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification of a mitochondrial gene, PET309, involved in C. auris fluconazole susceptibility using the PB-based mutagenesis system.

a Heatmap of the Pearson correlation coefficients (R) calculated across biological triplicates within and between screening conditions. b Volcano plots of the FSI values for PB transposition mutants grown in YPD containing 30, 60, or 120 μg/mL fluconazole compared with the same mutants grown in drug-free YPD. The PB-mutated genes represented by mutants that show statistically significant fold increases (FSI ≥ 1 and p < 0.05) or decreases (FSI ≤ 1 and p < 0.05) after drug treatment are indicated in red or blue, respectively. Three genes known to be associated with fluconazole susceptibility are highlighted in purple. The number of mutants that meet our selection criteria ( | FSI | ≥ 1, p < 0.05) is indicated on the top of each volcano plot. Statistical significance was tested using the Wald test with the Benjamini-Hochberg correction. c Spotting of the indicated strains on YNB plates supplemented with 2% glucose or 2% glycerol. Cells were grown overnight in YPD, washed twice with PBS, and resuspended in PBS at a final concentration of 1 × 107 cells/mL. Subsequently, 3 μL was spotted in serial 10-fold dilutions. Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. d Dose-response assays for the indicated strains were performed as described in Fig. 1f. Fluconazole was applied as 2-fold serail dilutions ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Averaged technical triplicates are represented by the color scale. The experiment was repeated twice with similar results.

Source data

Extended Data Fig. 2 Characterization of the screening strain, CauW156.

a Genotype description of CauW156. Arrows indicate primers used for genotyping. b PCR-based genotyping of CauW156 using primers shown in (a). WT and CauW08 cells were included as controls. The experiment was repeated twice with similar results. c Dose-response assays for the indicated strains were performed as described in Fig. 1f. Fluconazole was applied as 2-fold serial dilutions ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Averaged technical triplicates are represented by the color scale. The experiment was repeated twice with similar results. d Dose-response assays for the indicated strains were performed as described in Fig. 1f. Fluconazole was applied as 2-fold serial dilutions ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Averaged technical triplicates are represented by the color scale. The experiment was repeated twice with similar results.

Source data

Extended Data Fig. 3 Examination of the role of 000175 and its homologs in calcium and fluconazole susceptibility in C. auris, C. albicans, and S. cerevisiae.

a Spotting of the indicated strains on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 M CaCl2. Cells were grown overnight in YPD and then diluted to 1 × 107 cells/mL. Subsequently, 3 μL was spotted in 10-fold serial dilutions. Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. b Spotting assays for the indicated strains were performed on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 M CaCl2 as described in panel (a). Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. c Spotting assays for the indicated strains were performed on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 M CaCl2 as described in panel (a). Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. d Spotting assays for the indicated strains were performed on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 M CaCl2 or containing 5, 10, or 20 µg/mL fluconazole as described in panel b Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results.

Extended Data Fig. 4 Calcineurin promotes Cdt1’s calcium pump-independent function through a post-transcriptional mechanism.

a Calcium-binding loops in C. auris calmodulin, CMD1. Four potential calcium-binding loops were predicted based on the structure of S. cerevisiae calmodulin85. The residues highlighted in red were mutated (D to A and E to V) to abolish its calcium-binding activity, yielding CMD1-4. b Spotting assays for the indicated strains were performed on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 M CaCl2 as described in Extended Data Fig. 3a. Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. c Domain organization of the catalytic subunit of C. auris calcineurin, calcineurin A (CNA1). Catalytic, the phosphatase domain; CNB-binding, calcineurin B subunit domain; CAM-binding, calmodulin-binding domain; AI, the auto-inhibitory domain. The boundaries of these domains are defined according to the structure of the rat calcineurin A α-isoform70. Two stop codons were introduced at L453 to generate a constitutively activated calcineurin A, CNA1tr. d Dose-response assays for the indicated strains were performed as described in Fig. 3a. Fluconazole was applied as 2-fold serial dilution ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-way ANOVA multiple comparison test with the Geisser-Greenhouse correction. The experiment was repeated twice with similar results. e Corresponding MIC50 and SMG levels calculated from data in panel (d). MIC50 and SMG were calculated as described in Fig. 3b. f qPCR analysis of CDT1 transcript levels. The indicated strains were grown overnight in YPD and then subcultured at a 1:100 dilution into YPD containing 0 or 30 µg/mL fluconazole. Cells were collected for RNA extraction after 4 h of growth at 30 °C. CDT1 expression levels were normalized to ACT1, with the level of the untreated WT strain set to 1. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-tailed, unpaired Student’s t-test. The experiment was repeated twice with similar results. g Dose-response assays for the indicated strains were performed as described in Fig. 3a. Fluconazole was applied as 2-fold serial dilution ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-way ANOVA multiple comparison test with the Geisser-Greenhouse correction. The experiment was repeated twice with similar results. h Corresponding MIC50 and SMG levels calculated from data in panel (g). MIC50 and SMG were calculated as described in Fig. 3b. i The calcium-calcineurin signaling pathway drives Cdt1’s plasma membrane localization post-transcriptionally. The indicated strains expressing GFP-Cdt1 under the ADH1 promoter were grown overnight in YPD and then subcultured at a 1:100 dilution into YPD with 0 or 30 µg/mL fluconazole. Images were taken after 4 h of growth at 30 °C. Scale bar, 5 μm. The experiment was repeated twice with similar results.

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Extended Data Fig. 5 Cdt1’s calcium pump-independent function in fluconazole tolerance depends on its localization to the plasma membrane.

a Spotting assays for the indicated strains were performed on YPD plates supplemented with 0 or 0.4 M CaCl2 as described in Extended Data Fig. 3a. Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. b Effect of the K603E substitution in Cdt1 on its plasma membrane localization. The indicated strains expressing GFP-Cdt1 or GFP-Cdt1K603E were grown overnight in YPD and then subcultured at a 1:100 dilution into YPD with 0 or 30 µg/mL fluconazole. Images were taken after 4 h of growth at 30 °C. Scale bar, 5 μm. The experiment was repeated twice with similar results. c Quantification of the fluorescence intensity of GFP-Cdt1 at the plasma membrane observed in panel (b). Box plot format is the same as described in Fig. 3g. n = 50 for both WT and cdt1Δ:PCDT1-CDT1K603E cells. Statistical differences were determined by two-tailed, unpaired Student’s t-test. d Dose-response assays for the indicated strains were performed as in Fig. 3a. Fluconazole was applied as 2-fold serial dilutions ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-way ANOVA multiple comparison test with the Geisser-Greenhouse correction. The experiment was repeated twice with similar results. e Corresponding MIC50 and SMG levels calculated from data in panel (d). MIC50 and SMG were calculated as described in Fig. 3b.

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Extended Data Fig. 6 Sequence alignment of Cdt1 and its homologs using Clustal Omega (v.1.2.4).

From the top, the sequences of S. cerevisiae Pmc1, C. albicans Pmc1, and C. auris Cdt1. The conserved motif, DKTGTLT, around the phosphorylatable D429 (highlighted in red) is marked within a blue square.

Extended Data Fig. 7 Characterization of C. auris clinical isolates utilized in this study.

a Phylogenic analyses of 17 clinical strains isolated from hospitals in Beijing, China (marked in red), and four strains obtained from other labs are shown in blue. Fourteen representative isolates from all six C. auris clades are shown in black. The maximum-likelihood phylogenetic tree was generated based on WGS data. The scale bar represents the mean number of nucleotide substitutions per site. b Dose-response assays for the indicated strains were performed as described in Fig. 1f. Fluconazole was applied as 2-fold serial dilutions ranging from 0.5 to 512 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Averaged technical triplicates are represented by the color scale. The experiment was repeated twice with similar results. c Dose-response assays for the indicated strains were performed as described in Fig. 1f. Fluconazole was applied as 2-fold serial dilutions ranging from 1 to 1,024 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Data are presented as mean ± SD of technical triplicates. The experiment was repeated twice with similar results.

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Supplementary information

Supplementary Information

Extended Data figure legends, Supplementary Result, Fig. 1 and figure legend, and Tables 4–6.

Reporting Summary

Supplementary Table 1

Genome-wide profiling of genes that affect C. auris’s fluconazole susceptibility.

Supplementary Table 2

GO term enrichment analysis of cellular components represented in the PB-inserted genes associated with 58 concentration-independent mutants.

Supplementary Table 3

Differentially expressed genes (DEGs) in pet309Δ cells.

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Song, Y., Chen, J., Wan, J. et al. Candida auris vacuolar calcium pump mediates fluconazole efflux and resistance evolution. Nat Microbiol (2026). https://doi.org/10.1038/s41564-026-02270-1

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