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Cited by 31Fungal Infections and StudiesHeat shock proteins researchFungal and yeast genetics research

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence

Open access
Fu, Ci, Beattie, Sarah R, Jezewski, Andrew J, Robbins, Nicole, Whitesell, Luke, Krysan, Damian J, Cowen, Leah E·2022·Genetics
DOI 10.1093/genetics/iyab164PMC PMC8733452
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Abstract

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

AI snapshot

Written from the paper’s full text.

TL;DR

The authors generated a copper-repressible *CTR4-2p-HSP90* conditional mutant in *Cryptococcus neoformans* and used it to show that Hsp90 supports growth at host temperature, azole tolerance, stress responses, melanin production, and virulence in an inhalation mouse model. The work provides a genetic tool for studying this essential chaperone and proof-of-principle that Hsp90 is a promising antifungal target in cryptococcosis.

Key findings

  • Copper-mediated repression of *HSP90* reduced Hsp90 protein by 60%–70% relative to wild type at 30°C and 37°C, caused a severe growth defect at 37°C, and increased sensitivity to fluconazole but not amphotericin B.
  • Genetic or pharmacological Hsp90 compromise plus fluconazole caused an approximate 1 Log10 reduction in CFU after 24 h, indicating that Hsp90 inhibition enables fluconazole to kill *C. neoformans*.
  • Hsp90 depletion increased sensitivity to SDS, MG132, low pH, benomyl, hydroxyurea, and methyl methanesulfonate; caused abnormal polarized growth with multinucleated elongated cells; impaired melanin production at 37°C; and increased capsule thickness at 30°C in mutants while copper reduced capsule thickness in wild type.
  • In mice, the *CTR4-2p-HSP90* mutant showed attenuated virulence after intranasal inoculation (5 × 10^4 CFU) but no virulence defect after intravenous inoculation (1 × 10^4 CFU); *HSP90* expression was reduced in lung and increased in brain.

Methods

  • The *CTR4-2p-HSP90* mutant was constructed in KN99α by replacing 111 bp upstream of the *HSP90* intron with a NAT-*CTR4-2* promoter cassette using TRACE/CRISPR-Cas9; two PCR-validated transformants from a single transformation were characterized.
  • Phenotypic assays included spot dilution growth tests, E-test strips, fluconazole cidality CFU assays, propidium iodide microscopy, melanin production on dopamine medium, capsule measurement by India ink, and stress sensitivity panels.
  • Mouse virulence used 8-week-old female A/J mice (n = 10 per group) inoculated intranasally with 5 × 10^4 CFU or intravenously with 1 × 10^4 CFU; survival was analyzed by Kaplan-Meier curves with log-rank tests, and in vivo *HSP90* expression was measured by qRT-PCR normalized to *TEF1* (n = 3 mice per group).

Limitations

  • Stated: copper supplementation did not completely repress *HSP90* expression, and the authors note that alternative systems such as a tetracycline-repressible promoter might more tightly regulate *HSP90* and avoid copper-induced artifacts.
  • Stated: at 37°C, Hsp90 depletion caused a cytokinesis defect that prevented assessment of capsule production under capsule-inducing conditions.
  • Evident from the text: copper itself significantly reduced capsule thickness in wild-type cells, so copper-dependent effects may confound some phenotypes; residual Hsp90 in the mutant may also underestimate the full consequences of loss of function.

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

Integrity signals

Registry

Crossref · 10.1093/genetics/iyab164

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 63 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Abstract

    We generated a conditional expression strain of C. neoformans in which HSP90 is under control of the copper-repressible promoter CTR4-2.

    “we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2.”
    • Also stated in: § Results · plain
      “we constructed a conditional expression allele of HSP90 under control of the copper-suppressible promoter CTR4-2”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    In the CTR4-2p-HSP90 strain, supplementation with 25 μM CuSO4 significantly reduced HSP90 transcript levels at 37°C.

    “While supplementation with 25 µM CuSO4 had no significant effect on HSP90 expression in the wild-type control, it significantly reduced HSP90 expression in both transformants.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    Depletion of HSP90 increases sensitivity to benomyl, hydroxyurea, and methyl methanesulfonate at 37°C and 30°C.

    “Depletion of HSP90 increased sensitivity to all three compounds at 37°C (Figure 3A) and 30°C (Supplementary Figure S4A)”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    Depletion of HSP90 increases sensitivity to SDS, MG132, and low pH at both 37°C and 30°C.

    “an increase in sensitivity to SDS, MG132, and low pH was observed at both 37°C (Figure 3A) and 30°C (Supplementary Figure S3).”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    Addition of radicicol or HSP990 further exacerbates the growth defect of CTR4-2p-HSP90 strains in the presence of copper at both 37°C and 30°C.

    “Addition of the structurally unrelated Hsp90 inhibitors radicicol or HSP990 further exacerbated the growth defect phenotype of the CTR4-2p-HSP90 strains in the presence of copper at both temperatures (Figure 1D and Supplementary Figure S2B)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Radicicol and HSP990 specifically inhibit Hsp90 in C. neoformans.
  • May combine two claims. 17
  • Classified as probably not a substantial claim. 32
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data
    § Results

    Depletion of HSP90 causes abnormal polarized growth and multinucleate phenotypes in C. neoformans.

    “Closer examination of the cells via microscopy revealed abnormal polarized growth in the CTR4-2p-HSP90 mutants in the presence of copper (Figure 3C and Supplementary Figure S4B), with cells often displaying multinucleate phenotypes (Figure 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Copper-mediated repression of C. neoformans HSP90 causes a significant growth defect at 37°C and a marginal defect at 30°C.

    “Copper-mediated repression of C. neoformans HSP90 caused a significant growth defect at 37°C (Figure 1D) and a marginal defect at 30°C (Supplementary Figure S2B).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given
    § Discussion

    There is a very short intergenic sequence between HSP90 and YPD1.

    “there is a very short intergenic sequence between HSP90 and yet another essential gene, YPD1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    At 30°C, addition of copper significantly reduces capsule thickness of the wild-type strain but significantly increases capsule thickness in both CTR4-2p-HSP90 mutants.

    “at 30°C, addition of copper significantly reduced capsule thickness of the wild-type strain but caused a significant increase in capsule thickness in both CTR4-2p-HSP90 mutants (Figure 4, C and D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The reduction in melanin production by Hsp90 inhibition occurred at concentrations with minimal impact on growth, so it is not merely due to growth inhibition.

    “this occurred at concentrations that had minimal impact on growth, highlighting that the reduction in melanin production was not merely due to growth inhibition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Depletion of HSP90 in C. neoformans results in an apparent cytokinesis defect and the formation of multinucleated elongated cells.

    “we found that depletion of HSP90 in C. neoformans resulted in an apparent cytokinesis defect and the formation of multinucleated elongated cells”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given
    § Materials and methods

    All spot dilution experiments were performed in biological duplicate to confirm data reproducibility.

    “All experiments were performed in biological duplicate to confirm data reproducibility.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    Hsp90 modulates key virulence traits in C. albicans, such as morphogenesis, and genetic compromise of Hsp90 leads to clearance of otherwise lethal Candida or Aspergillus infections in animal models.

    “Hsp90 also modulates key virulence traits in C. albicans, such as the ability to undergo morphogenesis, and in animal models, genetic compromise of Hsp90 function leads to clearance of otherwise lethal Candida or Aspergillus infections (Cowen et al. 2009; Shapiro et al. 2009).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cowen et al. 2009

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    Study design and conductnone given
    § Materials and methods

    The plasmid was sequenced to confirm no mutations were introduced during the cloning steps.

    “The plasmid was sequenced to confirm no mutations were introduced during the cloning steps.”
    Quote in the paperNot a substantial claim

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    Premisecitation
    § Introduction

    C. neoformans can develop resistance to all current antifungals by employing genetic, genomic, and adaptive biological strategies.

    “C. neoformans is also able to develop resistance to all other current antifungals by employing a plethora of genetic, genomic, and adaptive biological strategies (Iyer et al. 2021).”
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    Study design and conductown data
    § Materials and methods

    In vivo gene expression primers were validated to amplify C. neoformans genes specifically, as all primer pairs tested failed to amplify cDNA from mock-infected tissues.

    “To ensure primers amplified C. neoformans genes specifically, we used cDNA from mock-infected mice administered PBS in lieu of fungal cells. All primer pairs tested failed to amplify cDNA from mock-infected tissues.”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § Introduction

    Targeting Hsp90 is a powerful therapeutic strategy to combat fungal disease caused by Candida and Aspergillus species.

    “Previous work has shown that targeting Hsp90 is a powerful therapeutic strategy to combat fungal disease caused by Candida and Aspergillus species (Cowen and Lindquist 2005; Cowen et al. 2009).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Cowen and Lindquist 2005 source

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    Interpretationown data
    § Results

    Hsp90 helps maintain protein homeostasis and genome stability in C. neoformans.

    “Hsp90 helps maintain protein homeostasis and genome stability”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § Introduction

    The copper-suppressible promoter CTR4-2 has been used previously to study function of the essential genes FAS1 and FAS2 in vivo.

    “the copper-suppressible promoter CTR4-2, which has been used previously to study function of the essential genes FAS1 and FAS2 in vivo (Chayakulkeeree et al. 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chayakulkeeree et al. 2007 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    Hsp90 inhibition abrogates antifungal drug resistance and potentiates antifungal activity in fungi, notably by impairing signaling through calcineurin and the Pkc1 cell wall integrity pathway.

    “Hsp90 inhibition abrogates antifungal drug resistance and potentiates antifungal activity through numerous mechanisms, most notably by impairing signaling through the protein phosphatase calcineurin and the Pkc1 cell wall integrity pathway (Cowen and Lindquist 2005; LaFayette et al. 2010; Singh-Babak et al. 2012; Caplan et al. 2018).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Cowen and Lindquist 2005 source

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    • cites LaFayette et al. 2010 source

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    Study design and conductcitation
    § Materials and methods

    The Hsp90 antibody recognizes two bands, and the top band has been validated as Hsp90 using synthesized C. neoformans Hsp90 protein.

    “The Hsp90 antibody recognizes two bands; the top band has been validated as Hsp90 using synthesized C. neoformans Hsp90 protein(Marcyk et al. 2021).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Marcyk et al. 2021 source

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    Study design and conductnone given
    § Materials and methods

    All western blot experiments were performed in biological duplicate to confirm data reproducibility.

    “All western blot experiments were performed in biological duplicate to confirm data reproducibility.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Results

    Melanin and capsule production are two major virulence traits in C. neoformans.

    “Melanin and capsule production are two major virulence traits in C. neoformans (Kwon-Chung et al. 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kwon-Chung et al. 2014 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    The molecular chaperone Hsp90 is conserved throughout eukaryotes and regulates diverse meta-stable signal transducers involved in sensing and responding to stress.

    “The molecular chaperone Hsp90 is conserved throughout eukaryotes, where it regulates the function of diverse meta-stable signal transducers involved in sensing and responding to stress (Taipale et al. 2010; O’Meara et al. 2017).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Taipale et al. 2010 source

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    Study design and conductown data
    § Materials and methods

    Transformants were genotyped via PCR for the correct integration of the CTR4-2 promoter.

    “Transformants were selected on YPD-NAT agar medium and genotyped via PCR for the correct integration of the CTR4-2 promoter.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given
    § Materials and methods

    The animal experiment protocols were approved by the Institutional Animal Care and Use Committee at the University of Iowa.

    “The animal experiment protocols were approved by Institutional Animal Care and Use Committee at the University of Iowa (Protocol: 0092064).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § Abstract

    In diverse fungal pathogens, Hsp90 governs fungal survival, drug resistance, and virulence.

    “In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cowen and Lindquist 2005 source

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    • cites Cowen et al. 2009

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    Premisecitation
    § Introduction

    C. neoformans is intrinsically resistant to echinocandins, and treatment options remain limited to polyenes, azoles, and flucytosine.

    “C. neoformans is intrinsically resistant to echinocandins, and therefore treatment options remain limited to polyenes, azoles, and the pyrimidine analog flucytosine that targets RNA and DNA synthesis (Denning 2003; Iyer et al. 2021).”
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    Interpretationcitation
    § Results

    The inhibition of capsule production by radicicol is likely the result of growth defects upon Hsp90 compromise in those conditions.

    “which is likely the result of growth defects upon Hsp90 compromise in those conditions”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § Results

    In C. albicans, genetic depletion of Hsp90 has provided proof-of-principle that targeting this chaperone has great therapeutic potential to treat systemic candidiasis.

    “In C. albicans, genetic depletion of Hsp90 has provided proof-of-principle that targeting this chaperone has great therapeutic potential to treat systemic candidiasis (Cowen et al. 2009)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cowen et al. 2009

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    Premisecitation
    § Discussion

    Essential genes required for survival serve as optimal targets for novel antifungal development.

    “Essential genes that are required for survival serve as optimal targets for novel antifungal development (Iyer et al. 2021).”
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    • cites Iyer et al. 2021 source

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    Premisecitation
    § Discussion

    In C. albicans, Hsp90 client proteins include regulators that play important roles in cell-cycle progression and morphogenesis.

    “In C. albicans, Hsp90 client proteins also include regulators that play important roles in cell-cycle progression and morphogenesis (Hossain et al. 2021).”
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    Premisecitation
    § Discussion

    Hsp90 is a critical stress-response regulator that has shown great promise as an antifungal target in Candida and Aspergillus.

    “Hsp90, a critical stress-response regulator that has shown great promise as an antifungal target in Candida and Aspergillus (Cowen and Lindquist 2005; Cowen et al. 2009).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cowen and Lindquist 2005 source

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    Premisecitation
    § Discussion

    Calcineurin inhibitors synergize with fluconazole against C. neoformans.

    “Calcineurin inhibitors also synergize with fluconazole against C. neoformans (Del Poeta et al. 2000)”
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    Premisecitation
    § Discussion

    In several fungal species, including S. cerevisiae and C. albicans, it is estimated that ∼20% of open reading frames are essential.

    “In several fungal species, including the model yeast Saccharomyces cerevisiae and the distantly related fungal pathogen C. albicans, it is estimated that ∼20% of the open reading frames in their genomes are essential (Winzeler et al. 1999; Segal et al. 2018).”
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    Premisecitation
    § Discussion

    In C. albicans, Hsp90 modulates cellular responses to antifungals by stabilizing components of the Pkc1-MAPK and calcineurin stress-response pathways.

    “In C. albicans, Hsp90 modulates cellular responses to antifungals by stabilizing components of the Pkc1-MAPK and calcineurin stress-response pathways (Cowen and Lindquist 2005; Cowen et al. 2009; Shapiro et al. 2009).”
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    • cites Cowen and Lindquist 2005 source

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    • cites Cowen et al. 2009

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    Premisecitation
    § Results

    Pharmacological inhibition of Hsp90 by radicicol in nutrient-limited capsule-inducing media inhibits capsule production.

    “previous reports that pharmacological inhibition of Hsp90 by radicicol in nutrient-limited capsule-inducing media inhibits capsule production”
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    • cites Cordeiro et al. 2016 source

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    Premisecitation
    § Discussion

    In C. neoformans, calcineurin governs both morphogenesis during sexual reproduction and growth at host temperature.

    “In C. neoformans, calcineurin governs both morphogenesis during sexual reproduction and growth at host temperature (Odom et al. 1997; Cruz et al. 2001).”
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    Premisecitation
    § Discussion

    Only a small number of essential genes have been identified and characterized in C. neoformans due to technical challenges of genetic manipulation.

    “only a small number of essential genes have been identified and characterized in C. neoformans due to the technical challenges of genetic manipulation in this organism (Ianiri and Idnurm 2015; Iyer et al. 2021).”
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    Premisecitation
    § Discussion

    Calcineurin is a conserved nonessential protein phosphatase critical for morphogenesis, drug resistance, and virulence in multiple Candida species.

    “Calcineurin is a conserved nonessential protein phosphatase critical for morphogenesis, drug resistance, and virulence in multiple Candida species (Cruz et al. 2002; Blankenship et al. 2003; Chen et al. 2011, 2012, 2014; Zhang et al. 2012).”
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    • cites Cruz et al. 2002 source

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    • cites Blankenship et al. 2003 source

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    • cites Chen et al. 2011, 2012, 2014; Zhang et al. 2012

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    Premisecitation
    § Discussion

    In C. albicans, Hsp90 modulates morphological transitions via the Ras1-PKA signaling pathway, as well as the cyclin-dependent kinase Pho85, cyclin Pcl1, and transcription factor Hms1.

    “In C. albicans, Hsp90 modulates morphological transitions via the Ras1-PKA signaling pathway, as well as the cyclin-dependent kinase Pho85, cyclin Pcl1, and transcription factor Hms1 (Shapiro et al. 2009, 2012).”
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    • cites Shapiro et al. 2009, 2012

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    Premisecitation
    § Discussion

    In S. cerevisiae, cell cycle is regulated by the oscillatory expression of stage-specific cyclins that bind to and activate the master CDK Cdc28.

    “In S. cerevisiae, cell cycle is regulated by the oscillatory expression of stage-specific cyclins that bind to and activate the master cyclin-dependent kinase (CDK) Cdc28 (Lew et al. 1997).”
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    • cites Lew et al. 1997

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    Premisecitation
    § Discussion

    The major pathway regulating titan cell formation is the cAMP/PKA/Rim101 signaling cascade.

    “The major pathway regulating titan cell formation is the cAMP/PKA/Rim101 signaling cascade (Dambuza et al. 2018; Hommel et al. 2018; Trevijano-Contador et al. 2018)”
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    Premisecitation
    § Discussion

    Unique to C. neoformans, the cell cycle is differentially regulated during the formation of polyploid titan cells, which are gigantic cells that form inside host lungs and play important roles in pathogenesis, stress responses, and drug tolerance.

    “Unique to C. neoformans, the cell cycle is differentially regulated during the formation of polyploid titan cells, which are gigantic cells that form inside host lungs and play important roles in pathogenesis, stress responses, and drug tolerance (Okagaki et al. 2010; Zaragoza et al. 2010).”
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    Premisecitation
    § Discussion

    In C. albicans, inhibition of Hsp90 destabilizes Cdc28 leading to cell cycle arrest and morphogenesis defects.

    “In C. albicans, inhibition of Hsp90 destabilizes Cdc28 leading to cell cycle arrest and morphogenesis defects (Senn et al. 2012).”
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    • cites Senn et al. 2012 source

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    Premisecitation
    § Discussion

    Many signal transducers in these pathways are regulated by Hsp90 in C. albicans.

    “many signal transducers in these pathways are regulated by Hsp90 in C. albicans (O’Meara et al. 2017).”
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    Premisecitation
    § Discussion

    Cell cycle regulators Pho85 and Pcl1 have been shown to be involved in the endoreplication pathway during auto-diploidization in C. neoformans.

    “Cell cycle regulators Pho85 and Pcl1 have also been shown to be involved in the endoreplication pathway during auto-diploidization in C. neoformans (Fu et al. 2021).”
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42 of 42

  • The paper relies on this without support.

    Copper concentrations are relatively high in lung compared to blood in mice during infection, and the inhalation and systemic models expose the pathogen to high and low copper respectively.

    “leveraging the fact that copper concentrations vary widely in different mouse tissues”
    “selected because they were expected to expose the strain to either high or low concentrations of copper during the initial phase of infection.”
    “the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood.”

  • The paper relies on this without support.

    Radicicol and HSP990 specifically inhibit Hsp90 in C. neoformans.

    “Addition of the structurally unrelated Hsp90 inhibitors radicicol or HSP990 further exacerbated the growth defect phenotype”
    “Both pharmacological inhibition of Hsp90 (Figure 2C) and genetic suppression of HSP90 (Figure 2D) caused an approximate one Log10 reduction”

  • The paper relies on this without support.

    Analogous experiments in C. neoformans have yet to be performed.

    “analogous experiments in C. neoformans have yet to be performed.”

  • The paper relies on this without support.

    By intranasal inoculation, the fungus must first establish infection in the lungs prior to lethal systemic dissemination.

    “By this route of administration, the fungus must first establish infection in the lungs prior to lethal systemic dissemination.”

  • The paper relies on this without support.

    By intravenous inoculation, C. neoformans rapidly disseminates directly to the brain.

    “By this route of administration, C. neoformans rapidly disseminates directly to the brain.”

  • The paper relies on this without support.

    The poor potency of human-targeted inhibitors against fungi coupled with the toxicity associated with inhibiting host Hsp90 have precluded these compounds from being tested as antifungals in vivo.

    “the poor potency of human-targeted inhibitors against fungi coupled with the toxicity associated with inhibiting host Hsp90 have precluded these compounds from being tested as antifungals in vivo.”

  • The paper relies on this without support.

    The human fungal pathogen C. neoformans poses a major burden on human health, with treatment options limited to only two major antifungal classes.

    “The human fungal pathogen C. neoformans poses a major burden on human health, with treatment options limited to only two major antifungal classes.”

  • The paper relies on this without support.

    Another key trait of fungal pathogens is the ability to develop antifungal drug tolerance and resistance.

    “Another key trait of fungal pathogens is the ability to develop antifungal drug tolerance and resistance.”
8 of 8

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?

  • Depletion of HSP90 increases susceptibility to azoles as well as other cellular stressors in C. neoformans.

    “Through the generation of a strain where levels of HSP90 could be repressed with a copper-repressible promoter, the authors showed depletion of HSP90 increases susceptibility to azoles as well as other cellular stressors in this pathogen79.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [79]

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.

  • Genotoxic-stress results assigned to two different Figure 3 panels

    Minor

    The text identifies Figure 3B as showing the tests with benomyl, hydroxyurea, and methyl methanesulfonate, but then cites Figure 3A for the 37°C results with those compounds.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Lew, 1997, The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology, 607

Cited by

Sorted by citations

Related

Authors
Fu, C., Beattie, S. R., Jezewski, A. J., Robbins, N., Whitesell, L., Krysan, D. J., & Cowen, L. E.
Affiliations
  • University of Toronto (CA)
  • University of Iowa (US)
Corresponding author
Cowen, Leah E
ORCID
Venue
Genetics
Publisher
Oxford University Press (OUP)
Published
2021-10-09
Volume
220
Issue
1
ISSN
  • 0016-6731 (ISSN-L)
  • 1943-2631
DOI
10.1093/genetics/iyab164
PMC
PMC8733452
OpenAlex
W3207753353
Semantic Scholar
e92ebc010876275a1ce11ef5d7308c1218239b71
Cited by
31
References
70
Citations by year
2026: 3 · 2025: 13 · 2024: 7 · 2023: 7 · 2022: 1
FWCI
3.96
Type
journal article
Language
en
Open access
Licence
academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
Locations
  • Genetics (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • F32 Postdoctoral Individual National Research Service Award (AI145160)
  • T32 NINDS Institutional Research Training Grant (5T32AI007260)
  • National Institute of Health (R01AI147541-01A1)
  • Canadian Institutes of Health Research (FDN-154288)
  • NIH R01 (R01AI120958)
  • Canada Research Chair
  • Microbial Genomics Infectious Disease
Topics
  • Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords
Cryptococcus neoformans, HSP90, molecular chaperones, conditional gene expression, fungal virulence, stress tolerance, melanogenesis, fungal morphology, drug resistance, antifungal drug targets
MeSH terms
Animals, Antifungal Agents, Copper, Cryptococcosis, Cryptococcus neoformans, Fungal Proteins, Stress, Physiological, Virulence, HSP90 Heat-Shock Proteins, Drug Resistance, Fungal, Mice

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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