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Cited by 56Fungal and yeast genetics researchAntifungal resistance and susceptibilityHeat shock proteins research

Functional Genomic Screening Reveals Core Modulators of Echinocandin Stress Responses in Candida albicans

Open access
Caplan, Tavia, Polvi, Elizabeth J., Xie, Jinglin L., Buckhalter, Shoshana, Leach, Michelle D., Robbins, Nicole, Cowen, Leah E.·2018·Cell Reports
DOI 10.1016/j.celrep.2018.04.084
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Abstract

Candida albicans is a leading cause of death due to fungal infection. Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis. Resistance is readily acquired via mutations in the drug target gene, FKS1. Both basal tolerance and resistance to echinocandins require cellular stress responses. We performed a systematic analysis of 3,030 C. albicans mutants to define circuitry governing cellular responses to echinocandins. We identified 16 genes for which deletion or transcriptional repression enhanced echinocandin susceptibility, including components of the Pkc1-MAPK signaling cascade. We discovered that the molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1, establishing key mechanisms through which Hsp90 mediates echinocandin resistance. We also discovered that perturbation of the CCT chaperonin complex causes enhanced echinocandin sensitivity, altered cell wall architecture, and aberrant septin localization. Thus, we provide insights into the mechanisms by which cellular chaperones enable crucial responses to echinocandin-induced stress.

AI snapshot

Written from the paper’s full text.

TL;DR

A functional genomic screen of 3,030 *Candida albicans* mutants identified 16 genes required for echinocandin tolerance, nine of which also enable Fks1-mediated resistance. Follow-up work showed that Hsp90 stabilizes the PKC pathway kinases Pkc1 and Bck1, and that the CCT chaperonin complex modulates echinocandin responses partly through actin folding and septin localization.

Key findings

  • Screening two mutant libraries covering ~45% of the genome (2,356 GRACE strains and 674 homozygous deletion mutants) yielded 16 genes whose loss of function enhanced caspofungin susceptibility; 15/16 also showed fitness defects with micafungin, and all but TSC11 and AHR1 were required for full proliferation with calcofluor white, while only Pkc1, Stt4, and Kex2 mutants were hypersensitive to fluconazole.
  • Nine of the tolerance modulators also enabled resistance: depletion of PKC1, GIN4, CCT8, TSC11, or RPS5 and deletion of SWI4, MKC1, STT4, or KEX2 each reduced caspofungin resistance of an FKS1^T199C clinical isolate by at least 2-fold, comparable to Hsp90 perturbation.
  • Hsp90 depletion reduced Pkc1 and Bck1 protein levels without significantly affecting their transcripts (p > 0.05), TAP-tagged Pkc1 co-purified with endogenous Hsp90 under basal and caspofungin-stress conditions, and Cdc37 depletion also reduced Pkc1 levels — establishing Pkc1 and Bck1 as Hsp90 client proteins.
  • Repression of each CCT subunit reduced echinocandin tolerance; CCT8 depletion increased calcofluor white (chitin) staining, which sorbitol reversed, did not alter aniline blue (glucan) staining, triggered Mkc1 phosphorylation, and mislocalized Cdc10-GFP in over 25% of cells.

Methods

  • Chemical-genetic screen: homozygous deletion strains grown ± 12 ng/mL caspofungin; GRACE strains grown with 0.05 μg/mL doxycycline ± 15 ng/mL caspofungin, with OD600 readouts; hits defined as <90% (GRACE) or <50% (deletion) growth relative to no-drug controls, then MIC assays performed.
  • Follow-up genetics: homozygous deletion or conditional expression strains constructed in an echinocandin-resistant FKS1^T199C homozygous mutant; susceptibility also tested against micafungin, fluconazole, calcofluor white, cytochalasin A, and benomyl.
  • Protein and cell biology: co-immunoprecipitation and western blotting (Hsp90, TAP, H3, phospho-p44/42 MAPK, PSTAIRE, tubulin antibodies), quantitative RT-PCR normalized to ACT1 or GPD1, and DIC/fluorescence microscopy with calcofluor white and aniline blue staining; statistics by one-way or two-way ANOVA in GraphPad Prism.

Limitations

  • The paper states no dedicated limitations section; the authors note that the CCT complex is conserved in mammalian cells, though computational analysis suggests substantial differences between the yeast and human CCT interactome.
  • Evident from the text: the libraries collectively cover only ~45% of the genome, so modulators outside the screened set would be missed; screening used single drug concentrations (12 and 15 ng/mL caspofungin), and phenotype verification for independently constructed mutants is reported only as "data not shown."
  • Evident from the text: the authors acknowledge the theoretical possibility that destabilizing one PKC cascade component upon Hsp90 depletion could ripple to others, though they argue this is inconsistent with prior findings that homozygous deletion of pathway components does not drastically alter Slt2/Mkc1 levels.

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

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2018.04.084

Cites

30 of 30 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 25 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § 1 ∙ Leah E. Cowen

    Nine of the modulators of echinocandin tolerance also enable echinocandin resistance.

    “Nine of the modulators of echinocandin tolerance also enable resistance”
    • Also stated in: § Introduction · plain
      “We identified 16 genes important for echinocandin tolerance and 9 genes that enable echinocandin resistance.”
    • Also stated in: § Discussion · boosted
      “We established that nine of the modulators of echinocandin tolerance also enable echinocandin resistance”
    Quote in the paperSubstantial claim
  • Findingown data
    § 1 ∙ Leah E. Cowen

    Hsp90 is required for the stability of Pkc1 and Bck1.

    “The molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1”
    • Also stated in: § 1 ∙ Leah E. Cowen · plain
      “We discovered that the molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1”
    • Also stated in: § Results · plain
      “reduction of Hsp90 levels by DOX-mediated transcriptional repression in the tetO-HSP90/hsp90Δ strain was accompanied by a reduction in both Pkc1 and Bck1 protein levels”
    Quote in the paperSubstantial claim
  • Findingown data
    § 1 ∙ Leah E. Cowen

    Screening 3,030 C. albicans mutants identified 16 genes that modulate echinocandin tolerance.

    “Screening 3,030 C. albicans mutants reveals 16 modulators of echinocandin tolerance”
    • Also stated in: § 1 ∙ Leah E. Cowen · plain
      “We identified 16 genes for which deletion or transcriptional repression enhanced echinocandin susceptibility”
    • Also stated in: § Introduction · plain
      “We identified 16 genes important for echinocandin tolerance and 9 genes that enable echinocandin resistance.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    Homozygous deletion of SWI4, MKC1, STT4, and KEX2 reduced caspofungin resistance of the clinical isolate by at least 2-fold.

    “Homozygous deletion of SWI4, MKC1, STT4, and KEX2 also reduced caspofungin resistance of the clinical isolate by at least 2-fold”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    The analysis unveiled nine genes not previously implicated in echinocandin tolerance, including DBP8, SMD3, NOP14, RPS5, SEC14, CCT8, KEX2, TSC11, and orf19.4205.1.

    “Our analysis also unveiled nine genes with diverse biological functions that had not previously been implicated in echinocandin tolerance, including those involved in rRNA processing (DBP8), mRNA splicing (SMD3), ribosome components or biogenesis (NOP14 and RPS5), phosphatidylinositol transfer (SEC14), protein folding and processing (CCT8 and KEX2), and those with unknown function (TSC11 and orf19”
    Quote in the paperSubstantial claim
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 13
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data
    § Results

    The screen identified 55 GRACE strains with growth reduced to <90% and 25 homozygous deletion strains with growth reduced to <50% in caspofungin relative to a no-drug control.

    “We identified 55 GRACE strains for which growth was reduced to <90% and 25 homozygous deletion strains for which growth was reduced to <50% in the presence of caspofungin relative to a no-drug control”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Eight GRACE strains displayed ≥4-fold hypersensitivity and eight homozygous deletion mutants displayed ≥8-fold hypersensitivity to caspofungin relative to wild-type.

    “We focused on eight GRACE strains displaying ≥4-fold hypersensitivity and eight homozygous deletion mutants displaying ≥8-fold hypersensitivity to caspofungin relative to their corresponding wild-type”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Mkc1 was phosphorylated in response to caspofungin and in response to depletion of CCT8.

    “Mkc1 was phosphorylated in response to caspofungin, as expected, and in response to depletion of CCT8”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § 1 ∙ Leah E. Cowen

    Both basal tolerance and resistance to echinocandins require cellular stress responses.

    “Both basal tolerance and resistance to echinocandins require cellular stress responses.”
    • Also stated in: § Introduction · plain
      “Stress response pathways are critical to enable C. albicans proliferation in the presence of echinocandins for strains that have not acquired specific adaptive mutations, a phenomenon referred to as tolerance, as well as for echinocandin-resistant strains harboring mutations in FKS1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Robbins et al., 2017 source

      Not checked in the free run.

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

    Premisecitation
    § 1 ∙ Leah E. Cowen

    Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis.

    “Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis.”
    • Also stated in: § Introduction · plain
      “The current first-line therapy for the treatment of systemic candidiasis is administration of the echinocandins, which disrupt cell wall integrity by targeting β-1,3-glucan synthase, encoded by FKS1”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Robbins et al., 2017 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Results

    Both Mkk2 and Mkc1 require Hsp90 for their stability.

    “both Mkk2 and Mkc1 require this chaperone for their stability”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Diezmann et al., 2012; LaFayette et al., 2010

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

    Premisecitation
    § Results

    In mammalian cells, Hsp90 physically interacts with the PKC isoenzyme ßII.

    “In mammalian cells, Hsp90 physically interacts with the PKC isoenzyme ßII”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gould et al., 2009 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § Results

    Depletion of the CCT complex results in actin perturbation, thereby inducing cell wall stress.

    “Our results support a model in which depletion of the CCT complex results in actin perturbation, thereby inducing cell wall stress.”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § Results

    In S. cerevisiae, all five septin subunits physically interact with the CCT complex.

    “in S. cerevisiae all five septin subunits physically interact with the CCT complex”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Dekker et al., 2008 source

      Not checked in the free run.

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

    Premisecitation
    § Results

    Cct8 is one of eight protein subunits of the CCT complex, which plays a role in the folding and assembly of cytoskeletal proteins.

    “Cct8 is one of eight protein subunits of the CCT complex, which plays a role in the folding and assembly of cytoskeletal proteins”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sternlicht et al., 1993 source

      Not checked in the free run.

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

    Premisecitation
    § Introduction

    Hsp90 stabilizes calcineurin and two of the four core components of the PKC pathway, Mkk2 and Mkc1.

    “Signaling through these pathways is modulated by the molecular chaperone Hsp90, which stabilizes calcineurin and two of the four core components of the PKC pathway, the mitogen-activated protein kinase (MAPK) kinase (MAPKK) Mkk2 and the MAPK Mkc1”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Diezmann et al., 2012; LaFayette et al., 2010; Singh et al., 2009

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data
    § Results

    Hsp90 and Cdc37 regulate PKC signaling, and Pkc1 and Bck1 are Hsp90 client proteins in C. albicans.

    “Thus, we establish mechanisms through which Hsp90 and Cdc37 regulate PKC signaling, and identify client proteins of Hsp90 in C. albicans.”
    Quote in the paperSubstantial claim

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

    Interpretationcitation
    § Discussion

    Our results are consistent with genetic interaction studies in S. cerevisiae that highlight genetic interactors with FKS1 span diverse biological processes.

    “Our results are also consistent with genetic interaction studies in S. cerevisiae that highlight genetic interactors with FKS1 span diverse biological processes (Costanzo et al., 2016”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Costanzo et al., 2016 source

      Not checked in the free run.

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

    Study design and conductown data
    § Discussion

    To quantify the percentage of cells with mislocalized Cdc10-GFP, 100 cells from each treatment were counted and the mean ± SD from two biological replicates was calculated.

    “To quantify the percentage of cells with mislocalized Cdc10-GFP, 100 cells from each treatment were counted and the mean ± SD from two biological replicates was calculated.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § Discussion

    Protein kinases are core transducers of cellular stress and a major class of Hsp90 client proteins.

    “Protein kinases are core transducers of cellular stress and a major class of Hsp90 client proteins”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Taipale et al., 2012 source

      Not checked in the free run.

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

    Interpretationreasoning
    § Discussion

    The identified genes may reflect a requirement for altered expression of proteins to compensate for inhibition of Fks1.

    “which may reflect a requirement for altered expression of proteins to compensate for inhibition of Fks1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    Septin mislocalization is either a result of or a response to cell wall stress induced by disruption of the CCT complex.

    “suggesting that septin mislocalization is either a result of or a response to cell wall stress induced by disruption of the CCT complex.”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § Discussion

    Hsp90 associates with more than one-half of the human kinome.

    “estimates suggesting that Hsp90 associates with more than one-half of the human kinome (Taipale et al., 2012”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Taipale et al., 2012 source

      Not checked in the free run.

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

    Premisecitation
    § Discussion

    Other studies identify only select kinases within a signaling cascade as Hsp90 clients.

    “This is in contrast to other studies that identify only select kinases within a signaling cascade as Hsp90 clients (Taipale et al., 2012”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Taipale et al., 2012 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    Fks1-mediated resistance has emerged in C. albicans.

    “Given the emergence of Fks1-mediated resistance in C. albicans”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Robbins et al., 2017 source

      Not checked in the free run.

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

    Premisecitation
    § Discussion

    The CCT interaction network includes over 136 genes involved in the nuclear pore complex, chromatin remodeling, and protein degradation in S. cerevisiae.

    “the CCT interaction network includes over 136 genes involved in the nuclear pore complex, chromatin remodeling, and protein degradation in S. cerevisiae (Dekker et al., 2008”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Dekker et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § Discussion

    Our discovery that the CCT complex governs both tolerance and resistance to echinocandins expands the repertoire of targets for therapeutic intervention.

    “Our discovery that the CCT complex governs both tolerance and resistance to echinocandins expands the repertoire of targets for therapeutic intervention.”
    Quote in the paperSubstantial claim
22 of 22

  • The paper relies on this without support.

    Much of the circuitry contributing to echinocandin tolerance and resistance remains unknown.

    “However, much of the circuitry contributing to echinocandin tolerance and resistance remains unknown.”

  • The paper relies on this without support.

    No physical interaction between Hsp90 and PKC has been identified in yeast.

    “however, no physical interaction has been identified in yeast”

  • The paper relies on this without support.

    Cdc37 facilitates Hsp90’s recognition of kinase client proteins.

    “as Cdc37 facilitates Hsp90’s recognition of kinase client proteins”

  • The paper relies on this without support.

    Cell wall homeostasis is integral to tolerance of cell wall stress.

    “Given that cell wall homeostasis is integral to tolerance of cell wall stress”
4 of 4

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?

  • In C. albicans, key client proteins of Hsp90 include calcineurin and multiple members of the PKC-MAPK cell wall integrity cascade (Pkc1, Bck1, Mkk2, and Mkc1).

    “In C. albicans, key client proteins of Hsp90 include the Ca2+-calmodulin-activated protein phosphatase calcineurin, and multiple members of the PKC-MAPK cell wall integrity cascade (Pkc1, Bck1, Mkk2 and Mkc1)75–78.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [77]

  • 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).”

    From Genetic Analysis of <i>Candida auris</i> Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance (2019) · cites this paper as [11]

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.

  • CCT is said to enable susceptibility rather than tolerance

    Minor

    The text says that repressing CCT subunits reduces echinocandin tolerance, but later says the CCT complex enables echinocandin susceptibility. Those statements assign opposite directions to CCT's effect.

1 of 1

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Related

Authors
Caplan, T., Polvi, E. J., Xie, J. L., Buckhalter, S., Leach, M. D., Robbins, N., & Cowen, L. E.
Affiliations
University of Toronto (CA)
Corresponding author
Cowen, Leah E.
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2018-05
Volume
23
Issue
8
Pages
2292-2298
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2018.04.084
OpenAlex
W2803225311
Semantic Scholar
8d6ad3b0043f570db937b41957c98bb5c727c3ed
Cited by
56
References
30
Citations by year
2026: 3 · 2025: 8 · 2024: 3 · 2023: 6 · 2022: 10 · 2021: 14 · 2020: 5 · 2019: 5 · 2018: 2
FWCI
2.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Canadian Institutes of Health Research (MOP-86452, MOP-119520, FDN-154288)
  • Natural Sciences and Engineering Research Council of Canada (06261, 462167)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
Keywords
Candida albicans, echinocandin resistance, FKS1, drug tolerance, cellular stress response, HSP90, cell wall biosynthesis, antifungal susceptibility, deletion mutants
MeSH terms
Candida albicans, Cell Wall, Fungal Proteins, Genes, Fungal, Stress, Physiological, Genomics, Drug Resistance, Fungal, Echinocandins, Septins

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