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LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris

Nature Microbiology volume 6, pages 842–851 (2021)Cite this article

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Abstract

The emergent fungal pathogen Candida auris exhibits high resistance to antifungal drugs and environmental stresses, impeding treatment and decontamination1,2,3. The fungal factors mediating this stress tolerance are largely unknown. In the present study, we performed piggyBac, transposon-mediated, genome-wide mutagenesis and genetic screening in C. auris, and identified a mutant that grew constitutively in the filamentous form. Mapping the transposon insertion site revealed the disruption of a long non-coding RNA, named DINOR for DNA damage-inducible non-coding RNA. Deletion of DINOR caused DNA damage and an upregulation of genes involved in morphogenesis, DNA damage and DNA replication. The DNA checkpoint kinase Rad53 was hyperphosphorylated in dinorΔ mutants, and deletion of RAD53 abolished DNA damage-induced filamentation. DNA-alkylating agents, which cause similar filamentous growth, induced DINOR expression, suggesting a role for DINOR in maintaining genome integrity. Upregulation of DINOR also occurred during exposure to the antifungal drugs caspofungin and amphotericin B, macrophages, H2O2 and sodium dodecylsulfate, indicating that DINOR orchestrates multiple stress responses. Consistently, dinorΔ mutants displayed increased sensitivity to these stresses and were attenuated for virulence in mice. Moreover, genome-wide genetic interaction studies revealed links between the function of DINOR and TOR signalling, an evolutionarily conserved pathway that regulates the stress response. Identification of the mechanism(s) by which DINOR regulates stress responses in C. auris may provide future opportunities for the development of therapeutics.

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Fig. 1: Identification of DINOR required for C. auris morphogenesis using PB mutagenesis.
Fig. 2: The filamentous growth of dinorΔ depends on Rad53-mediated pathways.
Fig. 3: Functional interactions of DINOR with 005430 and 000754.
Fig. 4: DINOR is required for the C. auris response to antifungal drug, H2O2 and SDS, and pathogenicity in mice.

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Data availability

The data supporting the findings of the present study are available within the paper and its Supplementary Information. All relevant data, including further image and processed data, are available by request from the corresponding authors. The PB library sequencing data are available under BioProject accession no. PRJNA679970. The RNA-seq data are deposited under Gene Expression Omnibus accession no. GSE171261. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Ministry of Science and Technology of China (grant no. 2018YFA0800200 to J.W.), National Natural Science Foundation of China (grant no. 21675098 to J.W.), THU-PKU Center for Life Sciences (J.G. and J.W.), National Medical Research Council of Singapore (grant nos. NMRC/OFIRG/0072/2018 and NMRC/OFIRG/0055/2019 to Y.W.) and National Research Foundation of Singapore (grant no. NRF-ISF003-3039/19 to Y.W.).

Author information

Authors and Affiliations

  1. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore

    Jiaxin Gao, Eve W. L. Chow, Xiaoli Xu & Yue Wang

  2. School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

    Jiaxin Gao, Chunling Cai, Yabing Song & Jianbin Wang

  3. Faculty of Health Sciences, University of Macau, Macau, China

    Haitao Wang

  4. Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    Jianbin Wang

  5. Chinese Institute for Brain Research, Beijing, China

    Jianbin Wang

  6. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Yue Wang

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Contributions

J.G., J.W. and Y.W. conceptualized and designed the study. J.G. and E.W.L.C. performed most experiments with assistance from C.C. for the RACE assay and X.X. for the macrophage and mouse-related experiments. H.W. and Y.S. contributed to the bioinformatics analyses. J.G. and Y.W. analysed the data and wrote the manuscript.

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Correspondence to Jiaxin Gao, Jianbin Wang or Yue Wang.

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Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Extended data

Extended Data Fig. 1 PB transposition in C. auris.

a, The PB system. White vertical lines indicate hyperactive mutations. The PB transposon cassette PB[LEU2] is integrated within ARG4. Arrows indicate the PCR primers used to detect PB excision. b, PB[LEU2] is stable at the ARG4 locus in CauW08 under non-inducing conditions. Gel electrophoresis of PCR products amplified from the genomic DNA of 24 colonies obtained from three independent experiments using primers shown in a. Wild-type (WT) cells were included as a control. c, Transposition efficiency. Error bars, s.d. from the mean of three independent experiments. d, Density curves of PB-specific site counts per 10 kb in the three libraries. e, Correlations of PB site counts per 10 kb between the three libraries.

Source data

Extended Data Fig. 2 Illustration of the procedures used for screening filamentous mutants using the PB mutagenesis system in C. auris.

See Methods for details. White arrowhead indicates a wrinkled colony.

Extended Data Fig. 3 Characterization of C. auris mutants that grew wrinkled colonies.

a, 004138:PB and 004138:LEU2 mutants. (Top) Genotype description. Arrows indicate primers used for genotyping. (Bottom left) Images show the colony morphologies of 004138:PB and 004138:LEU2 cells. Strains were grown on YPD agar at 30 °C for 3 days. (Bottom right) Images show the cellular morphologies of 004138:PB and 004138:LEU2 cells. Strains were grown in liquid YPD medium or on YPD agar at 30 °C for 18 h or 3 days before microscopy. Scale bar, 10 μm. b, 004734:PB and 004734:LEU2 mutants. Scale bar, 10 μm. The experiment was independently repeated three times with similar results. c, 003427:PB and 003427:LEU2 mutants. Scale bar, 10 μm. The experiment was independently repeated three times with similar results. d, P003879:PB and P003879:LEU2 mutants. Scale bar, 10 μm. The experiment was independently repeated three times with similar results.

Extended Data Fig. 4 PCR-based genotyping of the indicated strains using primers shown in Fig. 1c,d and Extended Data Fig. 3.

WT cells were included as controls. The experiment was independently repeated three times with similar results.

Source data

Extended Data Fig. 5 Identification of DINOR in C. auris.

a, qPCR analysis of 004840 and 004841 expression in WT and T004840:LEU2 cells. Overnight cultures of WT and T004840:LEU2 strains were collected for RNA isolation. The expression levels were normalized against GAPDH, and that of the WT sample was set as 1. Error bars represent s.d. from the mean of three independent experiments. b, Effect of 004840 and 004841 on the filamentous phenotype of T004840:LEU2 cells. (Right top) Description of genotypes of 004840:LEU2, 004841:LEU2, and 004840:SAT1 004841:LEU2 cells. Arrows indicate the primers used for genotyping. Bottom panel, Cellular and colony morphologies of 004840:LEU2, 004841:LEU2, and 004840:SAT1 004841:LEU2 cells. WT and T004840:LEU2 cells were included as controls. Scale bar, 10 μm. Right panel, Gel electrophoresis results of PCR-based genotyping of the indicated strains. WT cells were included as a control. The experiment was independently repeated three times with similar results. c, qPCR analysis of four non-overlapping regions downstream of 004840. qPCR templates derived from total RNA of WT cells with or without reverse transcription. ACT1 and GAPDH were included as controls. d, Detection of the potential transcript downstream of 004840 by primer-walking PCR. Top, Schematic description of primers (arrows) used for primer-walking PCR. Bottom, PCR templates were prepared from the total RNA of WT cells with or without the reverse transcription step. Genomic DNA was included as a control. The experiment was independently repeated three times with similar results.

Source data

Extended Data Fig. 6 DINOR functions as a lncRNA in C. auris morphogenesis.

a, Deleting DINOR led to filamentation. Images show the cellular and colony morphologies of dinorΔ and dinorΔ:DINOR cells. WT and T004840:LEU2 cells were included as controls. Scale bar, 10 μm. Columns on the right side show the percentages of yeast and filamentous cells of the indicated strains grown in liquid or solid YPD medium (n = 200). Error bars, s.d. from the mean of three independent experiments. b, Short ORFs (1-5) within DINOR identified using ORF Finder (http://www.ncbi.nlm.nih.gov/orffinder/). c, Expression of each putative ORF under the control of PENO1 failed to restore the yeast morphology of dinorΔ cells. Images show the cellular and colony morphologies of dinorΔ:PENO1-ORF1, dinorΔ:PENO1-ORF2, dinorΔ:PENO1-ORF3, dinorΔ:PENO1-ORF4, and dinorΔ:PENO1-ORF5 cells. WT and dinorΔ cells were included as controls. Scale bar, 10 μm. Columns at the bottom show the percentages of yeast and filamentous cells of the indicated strains grown in liquid or solid YPD medium (n = 200). Error bars, s.d. from the mean of three independent experiments. d, Mutating the start codon ATG of each ORF individually to AAG did not affect DINOR’s function. DINOR carrying the ATG-AAG mutation was integrated into the genome of dinorΔ. Images show the cellular and colony morphologies of dinorΔ:DINORT1115A, dinorΔ:DINORT1021A, dinorΔ:DINORT815A, dinorΔ:DINORT528A, and dinorΔ:DINORT319A cells. WT, dinorΔ, and dinorΔ:DINOR cells were included as controls. Scale bar, 10 μm. White vertical lines mark single nucleotide mutations. Columns at the bottom show the percentages of yeast and filamentous cells of the indicated strains grown in liquid or solid YPD medium (n = 200). Error bars, s.d. from the mean of three independent experiments.

Source data

Extended Data Fig. 7 Activation of Rad53-mediated DNA damage response in dinorΔ cells.

a, Hierarchical clustering of WT, dinorΔ, and dinorΔ:DINOR samples based on log2-transformed transcripts per kilobase million (log2TPM) values obtained by RNA-Seq analysis. b, qPCR analysis of selected differentially expressed genes in WT, dinorΔ, and dinorΔ:DINOR cells. The expression levels were normalized against GAPDH and expressed as relative quantity to the WT sample set as 1. Error bars, s.d. from the mean of three independent experiments. c, DNA degradation in dinorΔ cells. Overnight cultures of WT, dinorΔ, and dinorΔ:DINOR strains were collected for DNA extraction, and approximately 200 ng DNA of each strain was loaded for gel electrophoresis. The experiment was independently repeated three times with similar results. d, Morphologies of rad53Δ cells in the presence or absence of MMS. rad53Δ cells were treated with or without MMS at 30 °C for12 h. WT cells were included for comparison. Scale bar, 10 μm.

Source data

Extended Data Fig. 8 DINOR’s role in stress response is conserved across different clades of C. auris.

Antifungal drugs, H2O2, and SDS susceptibility assays for dinorΔ mutants derived from an India isolate (CBS12766; Clade I) were done as described in Fig. 4a.

Source data

Extended Data Fig. 9 Effect of sonication treatment on morphology and viability of WT, dinorΔ, and dinorΔ:DINOR cells.

a, Morphologies of WT, dinorΔ, and dinorΔ:DINOR cells after sonication. Cells were suspended in 20 ml PBS and sonicated three times, each for 20 s with 2 min cooling on ice between rounds. Sonication amplitude was set at 28%. Cells without treatment were included as controls. Scale bar, 10 μm. The experiment was independently repeated three times with similar results. b, Survival rates of WT, dinorΔ, and dinorΔ:DINOR cells after sonication. Cell survival was measured by counting CFUs and expressed as relative quantity to the untreated cells of each strain set as 100%. Error bars represent s.d. from the mean of three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

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Supplementary Video 1

Normal mice.

Supplementary Video 2

C. auris-infected mice.

Supplementary Data 1

DEGs in dinorΔ cells.

Supplementary Data 2

GO term enrichment analysis of DEGs in dinorΔ cells.

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Gao, J., Chow, E.W.L., Wang, H. et al. LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris. Nat Microbiol 6, 842–851 (2021). https://doi.org/10.1038/s41564-021-00915-x

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