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Cited by 136Antifungal resistance and susceptibilityFungal and yeast genetics researchFungal Infections and Studies

Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

Open access
Kim, Sang Hu, Iyer, Kali R., Pardeshi, Lakhansing, Muñoz, José F., Robbins, Nicole, Cuomo, Christina A., Wong, Koon Ho, Cowen, Leah E.·2019·mBio
DOI 10.1128/mbio.02529-18PMC PMC6355988
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Abstract

Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90 , which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90.

AI snapshot

Written from the paper’s full text.

TL;DR

This study adapted a tetracycline-repressible promoter system to show that Hsp90 is essential in *Candida auris*, mediates fluconazole tolerance in some clinical isolates, and represses filamentous growth. It also identifies the Cdr1 efflux transporter as a major contributor to azole resistance in *C. auris*.

Key findings

  • Hsp90 is essential in *C. auris*: doxycycline-mediated repression of tetO-HSP90 reduced HSP90 expression to less than 40% of wild-type and caused loss of viable colonies.
  • Hsp90 inhibition reduced fluconazole tolerance in *C. auris* clinical isolates CDC-382 and CDC-387 and transformed fluconazole activity from fungistatic to fungicidal, but did not affect high-level resistance in Ci6684 or CDC-388.
  • Deletion of CDR1 in strain Ci6684 caused an 8-fold decrease in fluconazole MIC, and CDR1 expression positively correlated with fluconazole MIC50 across clinical isolates (Pearson r = 0.59).
  • Hsp90 inhibition or depletion induced filamentous growth in *C. auris* and other CUG-clade species; RNA-Seq identified 2,111 *C. auris* DEGs upon geldanamycin treatment and 1,014 upon HSP90 depletion, with only 292 concordant DEGs.

Methods

  • A tetO-HSP90 conditional expression strain was constructed in fluconazole-resistant *C. auris* strain Ci6684 by CRISPR/Cas9-mediated promoter replacement, adapting a *C. albicans* system.
  • Drug susceptibility was assessed by spotting assays, fluconazole Etest strips, and checkerboard assays with fluconazole plus geldanamycin; CDR1 was deleted in Ci6684 and fluconazole MICs were measured.
  • Morphology was monitored by DIC microscopy after genetic depletion or pharmacological inhibition of Hsp90 and after canonical filament-inducing cues; RNA-Seq was performed in quadruplicate and analyzed with HISAT2, StringTie, and DESeq2.

Limitations

  • The paper states that the role of filamentous growth in *C. auris* is unclear, that only one prior study reported filamentation under high salt, and that further exploration of cues and genetic circuitry is needed.
  • Evident from the text: CDR1 deletion was tested only in the Ci6684 background, and Hsp90’s role in fluconazole tolerance was limited to a subset of CDC clinical isolates.
  • Evident from the text: RNA-Seq used strain Ci6684 but reads were aligned to the B8441 reference genome, and the study notes that depletion and pharmacological inhibition of Hsp90 produced distinct transcriptional responses.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1128/mbio.02529-18

Cites

53 of 53 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 40 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition.

    “Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling.”
    • Also stated in: § INTRODUCTION · plain
      “we discovered that genetic depletion or pharmacological inhibition of Hsp90 induced filamentous growth in C. auris.”
    • Also stated in: § Hsp90 represses filamentous growth in C. auris. · plain
      “During characterization of the C. auris tetO-HSP90 strain, we discovered that depletion of HSP90 resulted in polarized growth, similar to what was observed in C. albicans”
    Quote in the paperSubstantial claim
  • Findingown data

    Hsp90 inhibition in C. auris is accompanied by global transcriptional remodeling.

    “Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling.”
    • Also stated in: § C. auris filamentation is associated with transcriptional induction of putative cell surface genes. · plain
      “We identified global transcriptional changes in response to Hsp90 compromise in both species.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hsp90 is essential for growth in Candida auris.

    “We showed that Hsp90 is essential for growth in C. auris”
    • Also stated in: § HSP90 is essential in C. auris. · plain
      “Thus, Hsp90 is essential in C. auris, and the tetO promoter system offers a powerful approach for studying essential gene function in this emerging pathogen.”
    Quote in the paperSubstantial claim
  • Findingown data

    Only 744 DEGs in C. albicans showed concordant changes in expression under both Hsp90 depletion and inhibition.

    “Compared to Hsp90 depletion, only 744 DEGs in C. albicans showed concordant changes in expression under both conditions (Fig. 6A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Many of the genes ALS1, IFF4, RBT1, and KRE1 were also upregulated upon Hsp90 inhibition and depletion in C. albicans.

    “Many of these genes (ALS1, IFF4, RBT1, and KRE1) were also upregulated upon Hsp90 inhibition and depletion in C. albicans (Fig. 6C).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 19
  • Classified as probably not a substantial claim. 18

  • May combine two claims.

    Findingown data

    Depletion of HSP90 in C. auris led to upregulation of transcripts predicted to be involved in transmembrane transport and downregulation of genes annotated as involved in translation and peptide metabolic processes.

    “Depletion of HSP90 led to the upregulation of transcripts predicted to be involved in transmembrane transport and downregulation of genes annotated as involved in translation and peptide metabolic processes (Fig. S6 and Table S2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In total, 28 transcripts were upregulated and 17 transcripts were downregulated upon Hsp90 perturbation in both C. albicans and C. auris.

    “In total, 28 transcripts were upregulated and 17 transcripts were downregulated upon Hsp90 perturbation in both organisms (Fig. 6C).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    In C. albicans, filamentation caused by perturbations in Hsp90 is associated with upregulation of filament-associated genes such as HWP1, ECE1, and RBT1.

    “In C. albicans, filamentation caused by perturbations in Hsp90 was associated with upregulation of filament-associated genes such as HWP1, ECE1, and RBT1, consistent with previous reports (21, 41).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [21] Shapiro RS, Sellam A, Tebbji F, Whiteway M, Nantel A, Cowen LE 2012 Pho85, Pcl1, and Hms1 signaling governs Candida albicans morphogenesis induced by high temperature or Hsp90 compromise. Curr Biol 22: 461– 470. doi: 10.1016/j.cub.2012.01.062. 22365851 source

      Not checked in the free run.

    • cites [41] Braun BR, Head WS, Wang MX, Johnson AD 2000 Identification and characterization of TUP1-regulated genes in Candida albicans. Genetics 156: 31– 44. 10978273 PMC1461230 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    GO term enrichment analysis of DEGs in response to Hsp90 inhibition in C. auris identified upregulation of transcripts involved in ribosomal processes and downregulation of transcripts involved in metabolic and biosynthetic processes.

    “GO term enrichment analysis of the DEGs in response to Hsp90 inhibition identified upregulation of transcripts involved in ribosomal processes and downregulation of transcripts involved in metabolic and biosynthetic processes (Fig. S6 and Table S2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Of the C. albicans filamentous growth transcripts, 107 showed overlap under Hsp90 inhibition and depletion, including known regulators HWP1, ECE1, RBT1, BRG1, UME6, EFH1, and BCR1 upregulated and YWP1 downregulated.

    “Of these, 107 transcripts showed an overlap under the two conditions, including known regulators of filamentation HWP1, ECE1, RBT1, BRG1, UME6, EFH1, and BCR1, which were upregulated, and YWP1, which was downregulated (Fig. 6A; see also Table S1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Geldanamycin potentiated fluconazole activity against C. albicans clinical isolate CaCi2.

    “Although geldanamycin potentiated fluconazole activity against C. albicans, the combination had no effect on azole susceptibility in C. auris (Fig. 1C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    C. auris clinical isolates CDC-382 and CDC-387 do not contain any known Erg11 substitutions associated with azole resistance and show relatively low levels of CDR1 expression.

    “As CDC-382 and CDC-387 do not contain any known Erg11 substitutions associated with azole resistance and show relatively low levels of CDR1 expression, these two isolates may reflect a C. auris state prior to fluconazole exposure and acquisition of mutations associated with resistance (6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GO slim mapping of C. auris DEGs identified 79 and 33 transcripts involved in filamentous growth upon Hsp90 inhibition and depletion, respectively, but none overlapped with known regulators of C. albicans filamentation.

    “GO slim mapping of the DEGs identified 79 and 33 transcripts involved in filamentous growth upon Hsp90 inhibition and depletion, respectively, but none of these showed an overlap with the known regulators of C. albicans filamentation identified above (Fig. 6B; see also Table S3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Impairment of either Hsp90 function or cell cycle progression leads to filamentous growth in C. auris.

    “Here, we showed that the impairment of either Hsp90 function or cell cycle progression also leads to filamentous growth in this pathogen.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All species classified within the CUG clade showed filamentous growth in response to geldanamycin to various degrees, while non-CUG clade species remained exclusively in yeast form.

    “Interestingly, all species classified within the “CUG” clade showed filamentous growth in response to geldanamycin to various degrees, while the non-CUG clade species remained exclusively in yeast form (Fig. 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Depletion of HSP90 in C. albicans showed an enrichment profile distinct from pharmacological inhibition, with upregulation of oxidative stress response transcripts and downregulation of ribosomal and mitochondrial process transcripts.

    “Surprisingly, depletion of HSP90 showed a enrichment profile distinct from that observed upon pharmacological inhibition with Hsp90 in C. albicans, for which transcripts involved in responses to oxidative stress were upregulated and transcripts involved in ribosomal and mitochondrial processes were downregulated (see Fig. S5 and Table S2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Geldanamycin did not affect the high-level fluconazole resistance phenotypes of strain Ci6684 or strain CDC-388 or the fungistatic activity of fluconazole against these strains.

    “Notably, geldanamycin did not affect the high-level fluconazole resistance phenotypes of strain Ci6684 or strain CDC-388 or the fungistatic activity of fluconazole against these strains (Fig. 2B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Azole resistance mechanisms in C. albicans include alteration or overexpression of the target, activation or increased expression of azole efflux mediators, and induction of cellular stress responses.

    “Azole resistance mechanisms have been studied most extensively in C. albicans and include alteration or overexpression of the target, activation or increased expression of azole efflux mediators, and induction of cellular stress responses (7).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Robbins N, Caplan T, Cowen LE 2017 Molecular evolution of antifungal drug resistance. Annu Rev Microbiol 71: 753– 775. doi: 10.1146/annurev-micro-030117-020345. 28886681 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Azoles inhibit fungal growth by targeting lanosterol 14-alpha-demethylase (Erg11), a key component of ergosterol biosynthesis.

    “The most widely deployed class of antifungal is the azoles, which inhibit fungal growth by targeting lanosterol 14-α-demethylase (Erg11), a key component of the pathway for biosynthesis of the membrane sterol ergosterol (7).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Robbins N, Caplan T, Cowen LE 2017 Molecular evolution of antifungal drug resistance. Annu Rev Microbiol 71: 753– 775. doi: 10.1146/annurev-micro-030117-020345. 28886681 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. albicans, Hsp90 promotes the evolution of drug resistance by stabilizing regulators of cellular responses to drug-induced stress.

    “Specifically, in C. albicans, the molecular chaperone Hsp90 promotes the evolution of drug resistance by stabilizing regulators of cellular responses to drug-induced stress (8).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hsp90 represses filamentous growth in C. albicans such that compromising Hsp90 function induces the yeast-to-filament transition by relieving repression on Ras1-PKA signaling.

    “Hsp90 represses filamentous growth in C. albicans such that compromising Hsp90 function induces the yeast-to-filament transition by relieving repression on Ras1-protein kinase A (PKA) signaling (19).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, Heitman J, Cowen LE 2009 Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 19: 621– 629. doi: 10.1016/j.cub.2009.03.017. 19327993 PMC2735497 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Inhibition of Hsp90 blocks calcineurin-dependent stress responses and cell wall integrity signaling, reducing antifungal tolerance of clinical isolates and transforming azole activity from fungistatic to fungicidal.

    “Inhibition of Hsp90 blocks calcineurin-dependent stress responses and cell wall integrity signaling, thereby reducing antifungal tolerance of clinical isolates and transforming azole activity from fungistatic to fungicidal (8–11).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source

      Not checked in the free run.

    • cites [9] LaFayette SL, Collins C, Zaas AK, Schell WA, Betancourt-Quiroz M, Gunatilaka AAL, Perfect JR, Cowen LE 2010 PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90. PLoS Pathog 6: e1001069. doi: 10.1371/journal.ppat.1001069. 20865172 PMC2928802 source

      Not checked in the free run.

    • cites [10] Singh SD, Robbins N, Zaas AK, Schell WA, Perfect JR, Cowen LE 2009 Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog 5: e1000532. doi: 10.1371/journal.ppat.1000532. 19649312 PMC2712069 source

      Not checked in the free run.

    • cites [11] Caplan T, Polvi EJ, Xie JL, Buckhalter S, Leach MD, Robbins N, Cowen LE 2018 Functional genomic screening reveals core modulators of echinocandin stress responses in Candida albicans. Cell Rep 23: 2292– 2298. doi: 10.1016/j.celrep.2018.04.084. 29791841 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The ability of C. albicans to transition between yeast and filamentous forms is a key virulence trait triggered by environmental cues.

    “The ability of C. albicans to transition between yeast and filamentous forms is a key virulence trait that is triggered by a wide variety of environmental cues (20).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Shapiro RS, Robbins N, Cowen LE 2011 Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol Mol Biol Rev 75: 213– 267. doi: 10.1128/MMBR.00045-10. 21646428 PMC3122626 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. auris, point mutations in ERG11 contribute to azole resistance of clinical isolates.

    “In C. auris, studies have suggested that point mutations in ERG11 contribute to azole resistance of clinical isolates (15).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Healey KR, Kordalewska M, Jiménez Ortigosa C, Singh A, Berrío I, Chowdhary A, Perlin DS 2018 Limited ERG11 mutations identified in isolates of Candida auris directly contribute to reduced azole susceptibility. Antimicrob Agents Chemother 62: e01427-18. doi: 10.1128/AAC.01427-18. PMC6153782 30082281 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The impact of Hsp90 on drug resistance is conserved in other fungal pathogens, including Candida glabrata and Aspergillus fumigatus.

    “Moreover, the impact of Hsp90 on drug resistance has been conserved in a number of other fungal pathogens, including Candida glabrata and Aspergillus fumigatus (12–14).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [12] Cowen LE, Singh SD, Köhler JR, Collins C, Zaas AK, Schell WA, Aziz H, Mylonakis E, Perfect JR, Whitesell L, Lindquist S 2009 Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U S A 106: 2818– 2823. doi: 10.1073/pnas.0813394106. 19196973 PMC2650349 source

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    • cites [13] Singh-Babak SD, Babak T, Diezmann S, Hill JA, Xie JL, Chen Y-LL, Poutanen SM, Rennie RP, Heitman J, Cowen LE 2012 Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog 8: e1002718. doi: 10.1371/journal.ppat.1002718. 22615574 PMC3355103 source

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    • cites [14] Lamoth F, Juvvadi PR, Gehrke C, Steinbach WJ 2013 In vitro activity of calcineurin and heat shock protein 90 inhibitors against Aspergillus fumigatus azole- and echinocandin-resistant strains. Antimicrob Agents Chemother 57: 1035– 1039. doi: 10.1128/AAC.01857-12. 23165466 PMC3553695 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hsp90 governs temperature-dependent morphogenesis in C. albicans.

    “In addition to its role in enabling the emergence and maintenance of azole resistance, Hsp90 governs temperature-dependent morphogenesis in C. albicans (19).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, Heitman J, Cowen LE 2009 Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 19: 621– 629. doi: 10.1016/j.cub.2009.03.017. 19327993 PMC2735497 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Exposure to various cues that induce C. albicans filamentation does not induce morphogenesis in C. auris, suggesting divergence in the circuitry governing the yeast-to-filament transition between the two Candida species.

    “However, exposure to various cues that induce C. albicans filamentation does not induce morphogenesis in C. auris, suggesting divergence in the circuitry governing the yeast-to-filament transition between the two Candida species (23).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Larkin E, Hager C, Chandra J, Mukherjee PK, Retuerto M, Salem I, Long L, Isham N, Kovanda L, Borroto-Esoda K, Wring S, Angulo D, Ghannoum M 2017 The emerging pathogen Candida auris: growth phenotype, virulence factors, activity of antifungals, and effect of SCY-078, a novel glucan synthesis inhibitor, on growth morphology and biofilm formation. Antimicrob Agents Chemother 61: e02396-16. doi: 10.1128/AAC.02396-16. 28223375 PMC5404565 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Early C. albicans clinical isolates from an AIDS patient showed an Hsp90-dependent fluconazole tolerance phenotype.

    “The fluconazole-potentiating effect of Hsp90 inhibition is reminiscent of the results of a study of a series of C. albicans clinical isolates from an AIDS patient, with the early clinical isolates showing an Hsp90-dependent fluconazole tolerance phenotype (8, 27, 28).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source

      Not checked in the free run.

    • cites [27] White TC 1997 Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41: 1482– 1487. 9210670 PMC163944 source

      Not checked in the free run.

    • cites [28] Hill JA, O’Meara TR, Cowen LE 2015 Fitness trade-offs associated with the evolution of resistance to antifungal drug combinations. Cell Rep 10: 809– 819. doi: 10.1016/j.celrep.2015.01.009. 25660029 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Only one study has reported filamentous growth in C. auris to date, and that growth occurred under conditions of high salt concentrations.

    “Only one study has reported filamentous growth in C. auris to date, and that growth occurred under conditions of high salt concentrations (31).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Wang X, Bing J, Zheng Q, Zhang F, Liu J, Yue H, Tao L, Du H, Wang Y, Wang H, Huang G 2018 The first isolate of Candida auris in China: clinical and biological aspects. Emerg Microbes Infect 7: 93. doi: 10.1038/s41426-018-0095-0. 29777096 PMC5959928 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In C. albicans isolates from an AIDS patient, fluconazole resistance increased stepwise and became independent of Hsp90 as the isolates acquired mutations in ERG11 and TAC1.

    “Over time, these isolates showed stepwise increases in fluconazole resistance that became independent of Hsp90 as the isolates acquired mutations in ERG11 and in the transcriptional activator of CDR1, TAC1 (8, 27, 28).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source

      Not checked in the free run.

    • cites [27] White TC 1997 Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41: 1482– 1487. 9210670 PMC163944 source

      Not checked in the free run.

    • cites [28] Hill JA, O’Meara TR, Cowen LE 2015 Fitness trade-offs associated with the evolution of resistance to antifungal drug combinations. Cell Rep 10: 809– 819. doi: 10.1016/j.celrep.2015.01.009. 25660029 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cell surface proteins are often important regulators of morphogenesis and many are required for flocculation and adhesion.

    “Although universal changes in cell surface composition upon morphogenesis have not been identified in fungi, cell surface proteins are often important regulators of morphogenesis and many are required for flocculation and adhesion, which are phenotypes associated with specific morphogenetic states (43).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Gow NAR, Latge J-P, Munro CA 2017 The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr 5. doi: 10.1128/microbiolspec.FUNK-0035-2016. PMC11687499 28513415 source

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  • May combine two claims.

    Interpretationown data

    The tetracycline-repressible promoter system can be used to regulate expression of HSP90 in C. auris and establishes a precedent for genetic analysis of essential genes in this pathogen.

    “Our use of the tetracycline-repressible promoter system to regulate the expression of HSP90 in C. auris establishes a precedent for genetic analysis of essential genes in this pathogen.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Morphogenesis is an important virulence trait in C. albicans and other fungal pathogens.

    “Although morphogenesis is an important virulence trait in C. albicans and other fungal pathogens (40), the role of filamentous growth in C. auris is unclear.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Gow NAR, Brown AJP, Odds FC 2002 Fungal morphogenesis and host invasion. Curr Opin Microbiol 5: 366– 371. 12160854 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationown data

    A core transcriptional response accompanies C. albicans morphogenesis in response to perturbation of Hsp90 function.

    “This analysis validates the idea that a core transcriptional response accompanies C. albicans morphogenesis in response to perturbation of Hsp90 function and provides a powerful platform for comparative analyses with the transcriptional response in C. auris.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Antifungal exposure elicits distinct transcriptional responses between C. albicans and C. auris.

    “In addition, antifungal exposure has been shown to elicit distinct transcriptional responses between C. albicans and C. auris, suggesting that distinct genes may contribute to antifungal resistance in C. auris (18).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Muñoz JF, Gade L, Chow NA, Loparev VN, Juieng P, Berkow EL, Farrer RA, Litvintseva AP, Cuomo CA 2018 Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species. Nat Commun 9: 5346. 30559369 PMC6297351 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. albicans, filamentous growth is associated with extensive cell surface remodeling at the transcriptomic and proteomic levels.

    “In C. albicans, filamentous growth is also associated with extensive cell surface remodeling as indicated by changes in cell surface components at the transcriptomic and proteomic levels (32).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, Bouin A-PP, Sensen CW, Hogues H, van Het Hoog M, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M, Van Het HM, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M 2002 Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13: 3452– 3465. doi: 10.1091/mbc.e02-05-0272. 12388749 PMC129958 source

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  • May combine two claims.

    Interpretationown data

    Although the specific genes regulated during the morphogenetic transition are largely distinct between C. auris and C. albicans, the regulation of cell surface-associated genes during filamentous growth appears conserved between the two species.

    “Although the specific genes that are regulated during the morphogenetic transition are largely distinct between the two species, the regulation of cell surface-associated genes during filamentous growth appears to be conserved between C. auris and C. albicans.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Azole resistance in C. auris strains with ERG11 mutations and high CDR1 expression is likely due to poor engagement of fluconazole with its cellular target Erg11 and/or to upregulation of azole efflux pumps.

    “This highlights that azole resistance in these strains is likely due to the poor engagement of fluconazole with its cellular target Erg11 and/or to the upregulation of azole efflux pumps, thereby minimizing drug-induced cellular stress.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Plasmids used in this study had no nonsynonymous mutations, as verified by sequencing.

    “The absence of nonsynonymous mutations in plasmids was verified by sequencing.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    RNA for RNA-Seq was prepared in quadruplicate.

    “RNA was prepared in quadruplicate as described above.”
    Quote in the paperNot a substantial claim
35 of 35

  • The paper relies on this without support.

    Little is known about the mechanisms of azole resistance in C. auris, and genetic control of azole resistance in this organism has yet to be explored.

    “However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored.”
    “Despite the high incidence of azole resistance reported for C. auris isolates, little is known about the mechanisms involved.”
    “Despite these initial reports, genetic control of azole resistance has yet to be explored in C. auris.”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Cdr1, Mdr1 and Pdr16 cause clinical azole resistance.

    “including the Cdr1 multidrug efflux ATP-binding cassette (ABC) transporter, the Mdr1 major facilitator and the putative phosphatidylinositol transfer protein Pdr16 (ref. 18), all of which cause clinical azole resistance16,19,20.”

    From Candida auris skin tropism and antifungal resistance are mediated by carbonic anhydrase Nce103 (2025) · cites this paper as [20]

    The Results section, “Hsp90-independent fluconazole resistance is mediated in part by Cdr1” (page not provided), reports that deleting CDR1 reduced the fluconazole MIC eightfold in a C. auris isolate, supporting Cdr1’s role in azole resistance. The cited passages do not establish that Mdr1 or Pdr16 cause clinical azole resistance. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Some genes expressed in C. albicans hyphal phase have no orthologs in C. auris or no significant change of expression in different conditions inducing C. auris filamentation.

    “Moreover, some genes expressed in C. albicans hyphal phase have no orthologs in C. auris (e.g., ECE1, HWP1) or no significant change of expression in different conditions inducing C. auris filamentation (e.g., FLO8, EFG1, BRG1, BCR1, EFH1), suggesting distinct pathways between these genotypically distant Candida spp. (20, 37, 47).”

    From Ume6-dependent pathways of morphogenesis and biofilm formation in <i>Candida auris</i> (2024) · cites this paper as [37]

    The Results section (page unavailable) states that C. auris lacks orthologs of HWP1 and ECE1. It also reports that, under Hsp90 inhibition and depletion, its differentially expressed filamentous-growth genes did not overlap with the known C. albicans regulators examined. The copy does not establish the stated expression result for each of FLO8, EFG1, BRG1, BCR1 and EFH1 across different conditions that induce C. auris filamentation. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • PHR1 is upregulated following Hsp90 repression/inhibition, along with SCF1 and other genes of cell surface proteins exhibiting increased expression in UME6HA.

    “PHR1 was also upregulated following Hsp90 repression/inhibition, along with SCF1 and other genes of cell surface proteins exhibiting increased expression in UME6HA (PGA26, RBT4/KRE1) (37).”

    From Ume6-dependent pathways of morphogenesis and biofilm formation in <i>Candida auris</i> (2024) · cites this paper as [37]

    The Results section (page not available) reports that PHR1, PGA26, RBT1, and KRE1 were upregulated during C. auris filamentation associated with Hsp90 perturbation. It does not establish that PHR1 increased under both Hsp90 repression and inhibition, and the passages do not support the additional claims about SCF1 or increased expression in UME6HA. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Conditions favoring the filamentous phase of C. auris, such as blood contact and Hsp90 deprivation, share upregulated genes with UME6HA.

    “Together with other data sets containing genes upregulated in contact with blood cells [node “BLOOD_60 MIN_UP” (36)] and genes upregulated by deprivation of Hsp90 [node “HSP90_TET_UP”, (37)], these conditions have in common that they favor the filamentous phase of C. auris, which is consistent with the morphological effect of UME6HA on C. auris.”

    From Ume6-dependent pathways of morphogenesis and biofilm formation in <i>Candida auris</i> (2024) · cites this paper as [37]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • CDC-387 is both excluded from and described as showing fluconazole tolerance

    Minor

    The first passage identifies CDC-381 and CDC-382, but not CDC-387, as the tolerant isolates among the three more susceptible strains. The later passage describes geldanamycin as abolishing CDC-387's fluconazole tolerance.

  • Two different gene identifiers are assigned to the C. auris PHR1 ortholog

    Minor

    The Results and Discussion assign different gene identifiers to PHR1, leaving the identity of the reported upregulated gene inconsistent.

2 of 2

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Authors
Kim, S. H., Iyer, K. R., Pardeshi, L., Muñoz, J. F., Robbins, N., Cuomo, C. A., Wong, K. H., & Cowen, L. E.
Affiliations
  • University of Toronto (CA)
  • University of Macau (MO)
  • Broad Institute (US)
Corresponding author
Cowen, Leah E.
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2019-02-26
Volume
10
Issue
1
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.02529-18
PMC
PMC6355988
OpenAlex
W2912190166
Semantic Scholar
faa753073db5582ce1a9af26d79179b9584623e5
Cited by
136
References
53
Citations by year
2026: 15 · 2025: 13 · 2024: 18 · 2023: 17 · 2022: 18 · 2021: 21 · 2020: 25 · 2019: 9
FWCI
13.98
Type
journal article
Language
en
Update notices
correction (2019-03-19)
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Canadian Institutes of Health Research (FDN154288, 119520_1, MOP-86452)
  • National Institute of Allergy and Infectious Diseases (U19AI110818)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
Keywords
Candida auris, HSP90, azole resistance, yeast-to-hypha transition, morphogenesis, conditional gene expression, ergosterol biosynthesis, antifungal resistance, virulence, transcriptional remodeling, fungal pathogens
MeSH terms
Antifungal Agents, Azoles, Candida, Drug Tolerance, Gene Deletion, HSP90 Heat-Shock Proteins, Drug Resistance, Fungal, Membrane Transport Proteins, Gene Knockdown Techniques

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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