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Cited by 57Fungal Infections and StudiesHeat shock proteins researchFungal and yeast genetics research

Heat shock protein 90 localizes to the surface and augments virulence factors of Cryptococcus neoformans

Open access
Chatterjee, Sharanya, Tatu, Utpal·2017·PLOS Neglected Tropical Diseases
DOI 10.1371/journal.pntd.0005836PMC PMC5559104
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Abstract

BACKGROUND: Thermotolerance is an essential attribute for pathogenesis of Cryptococcus as exemplified by the fact that only two species in the genus, which can grow at 37°C, are human pathogens. Species which have other virulence factors including capsule formation and melanisation, but lack the ability to propagate at 37°C are not pathogenic. In another related fungal pathogen, Candida albicans, heat shock protein 90 has been implicated to be a central player in commanding pathogenicity by governing yeast to hyphal transition and drug resistance. Exploring Hsp90 biology in Cryptococcus in context of thermotolerance may thus highlight important regulatory principles of virulence and open new therapeutic avenues. METHODOLOGY/PRINCIPAL FINDINGS: Hsp90 is involved in regulating thermotolerance in Cryptococcus as indicated by growth hypersensitivity at 37°C upon mild compromise of Hsp90 function relative to 25°C. Biochemical studies revealed a more potent inhibition of ATPase activity by pharmacological inhibitor 17-AAG at 37°C as compared to 25°C. Catalytic efficiency of the protein at 37°C was found to be 6.39×10-5μM-1. Furthermore, indirect immunofluorescence analysis using a specific antibody revealed cell surface localization of Hsp90 via ER Golgi classical secretory pathway. Hsp90 was found to be induced under capsule inducing conditions and Hsp90 inhibition led to decrease in capsular volume. Finally compromising Hsp90 function improved anidulafungin tolerance in Cryptococcus. CONCLUSIONS/SIGNIFICANCE: Our findings highlight that Hsp90 regulates pathogenicity of the fungus by myriad ways. Firstly, it is involved in mediating thermotolerance which implies targeting Hsp90 can abrogate thermotolerance and hence growth of the fungus. Secondly, this study provides the first report of biochemical properties of Hsp90 of a pathogenic fungus. Finally, since Hsp90 is localised at the cell wall, targeting cell surface Hsp90 can represent a novel strategy to combat this lethal infection.

AI snapshot

Written from the paper’s full text.

TL;DR

Hsp90 is required for thermotolerance in *Cryptococcus neoformans*, localizes to the fungal cell surface via the ER–Golgi secretory pathway, and contributes to capsule regulation and intrinsic anidulafungin tolerance. The study reports the first biochemical characterization of Hsp90 from a pathogenic fungus and suggests cell-surface Hsp90 as a potential antifungal target.

Key findings

  • Hsp90 inhibition by radicicol was much more potent at 37°C than 25°C: MIC50 was 15.67 μM at 25°C versus 1.78 μM at 37°C for *C. neoformans* MTCC 1353, and 18.63 μM versus 1.51 μM for a clinical isolate; *C. albicans* MIC values remained the same at both temperatures.
  • Recombinant CnHsp90 was a functional ATPase: ATP Kd = 497.05 μM; Km = 324.95 μM at 25°C and 526.85 μM at 37°C; catalytic efficiency = 5.07 × 10−5 min−1 μM−1 at 25°C and 6.39 × 10−5 min−1 μM−1 at 37°C. 17-AAG inhibited ATPase activity more potently at 37°C (IC50 117.15 μM at 25°C versus 26.89 μM at 37°C).
  • Hsp90 localized to the cell surface at 25°C and 37°C, co-localized with calcofluor white, and was detected by whole-cell ELISA and β-mercaptoethanol surface extraction; BFA, NEM, and radicicol abolished surface localization.
  • Hsp90 was upregulated under capsule-inducing conditions, and radicicol treatment before or after capsule induction reduced capsular volume by approximately 60%. Hsp90 inhibition plus anidulafungin at 37°C reduced growth by about 42% at 0.25 μM radicicol and 47% at 0.5 μM radicicol, with ∑FIC = 0.56 indicating additivity; no effect was seen at 25°C.

Methods

  • Used *C. neoformans* strain MTCC 1353 and a clinical isolate, grown in YPD or Sabouraud dextrose broth; drug susceptibility was measured by broth microdilution MIC assays with radicicol and anidulafungin, plus spotting assays, immunoblotting, whole-cell ELISA, indirect immunofluorescence without permeabilization, and capsule induction in 10% fetal bovine serum.
  • Recombinant 6×-His-tagged CnHsp90 was cloned from *C. neoformans* cDNA, expressed in *E. coli*, and purified by Ni-NTA chromatography; ligand binding was measured by tryptophan fluorescence quenching, and ATPase activity was measured with γ-32P-ATP and thin-layer chromatography at 25°C and 37°C.
  • Data were analyzed with GraphPad Prism 5.0 using non-linear regression, Michaelis–Menten kinetics, one-way ANOVA, and two-tailed paired tests; capsule volume was calculated from measured whole-cell and cell-body diameters for 40 cells per experiment on at least three separate occasions.

Limitations

  • The paper does not include a dedicated limitations section. It states that in vitro ATPase IC50 differences are not sufficient alone to explain cellular-level effects because in vivo Hsp90 ATPase activity is regulated by cochaperones such as Sba1p and Aha1.
  • Evident from the text: the biochemical studies use recombinant CnHsp90 purified from *E. coli*, not native fungal Hsp90, and several mechanistic claims about client proteins, Hog1/PKC/PKA signaling, and calcineurin are presented as speculation rather than direct experimental validation.
  • Evident from the text: surface localization relies on antibody-based assays, and the drug-combination and capsule experiments are limited to the tested strain/clinical isolate and conditions; no animal infection model or in vivo virulence data are reported.

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

Integrity signals

Registry

Crossref · 10.1371/journal.pntd.0005836

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 36 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Hsp90 is significantly upregulated under 37°C and 5% CO2.

    “We also observed significant upregulation of Hsp90 under another well-established capsule inducing condition i.e. 37°C and 5% CO2 which mimics lung environment (S1B Fig).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.
  • Findingown data

    Hsp90 is localised on the fungal cell surface at both 25°C and 37°C.

    “Hsp90 was observed to be localised on the fungal cell surface at both 25°C and 37°C.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hsp90 inhibition with RAD during capsule induction leads to approximately 60% reduction in capsular volume compared to no drug.

    “Significant reduction in capsule size was observed when RAD was added in the capsule inducing media as compared to no drug (Fig 6C).”
    • Also stated in: § Hsp90 regulates capsule induction and maintenance in C. neoformans · plain
      “treatment with RAD during capsule induction led to approximately 60% reduction in capsular volume (Fig 6D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.
  • Findingown data

    RAD treatment (0.5 μM for 48 h) leads to loss of cell surface Hsp90 signal at both 25°C and 37°C.

    “Signal at the cell surface for Hsp90 was not observed at both temperatures (Fig 5D).”
    • Also stated in: § Cell surface association of Hsp90 depends on ER Golgi secretory pathway · hedged
      “Loss of cell surface localization was seen in case of RAD treated cells indicating pharmacological inhibitor can also interfere with the process of Hsp90 transport to cell surface.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hsp90 is markedly upregulated under capsule-inducing conditions (10% serum).

    “We probed protein levels under capsule inducing conditions and found a marked upregulation of Hsp90 under these conditions (Fig 6B).”
    • Also stated in: § Hsp90 regulates capsule induction and maintenance in C. neoformans · plain
      “Hsp90 is significantly upregulated under capsule inducing conditions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.
  • May combine two claims. 35
  • The quote only partly states this claim. 3
  • Classified as probably not a substantial claim. 20

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

    Findingown data

    C. neoformans is resistant to anidulafungin (AF) at both 25°C and 37°C, with normal growth even at 16 μg/ml.

    “We observed robust resistance to AF as indicated by normal growth of cells even at high doses of AF (16μg/ml).”
    • Also stated in: § Hsp90 governs echinocandin resistance at 37°C but not at 25°C · plain
      “Therefore, C. neoformans is resistant to AF and the resistance profile is similar at 25°C and 37°C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Catalytic efficiency of CnHsp90 was 5.07 × 10−5 min−1 μM−1 at 25°C and 6.39 × 10−5 min−1 μM−1 at 37°C.

    “The catalytic efficiency of CnHsp90 was found to be 5.07 × 10−5 min−1 μM−1 at 25°C and 6.39 × 10−5 min−1 μM−1 at 37°C (Fig 2E).”
    • Also stated in: § Methodology/Principal findings · plain
      “Catalytic efficiency of the protein at 37°C was found to be 6.39×10−5μM-1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    C. albicans growth is not hypersensitive to Hsp90 inhibition at 37°C; MIC values remain the same at both temperatures tested.

    “We did the same growth inhibition assay in presence of RAD with C. albicans as an organismal control wherein we found that MIC values remain the same at both temperatures tested (Fig 1F).”
    Quote in the paperSubstantial claim

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

    Findingown data

    CnHsp90 shows 69% identity with C. albicans Hsp90 and 68% identity with human Hsp90.

    “At the level of primary structure, C. neoformans Hsp90 shows 69% and 68% identity with C. albicans and human Hsp90 respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CnHsp90 obeys Michaelis-Menten kinetics; Km for ATP hydrolysis was 324.95 μM at 25°C and 526.85 μM at 37°C.

    “CnHsp90 obeys Michaelis Menten kinetics and Km for ATP hydrolysis for CnHsp90 were found to be 324.95 μM and 526.85 μM respectively at 25°C and 37°C (Fig 2D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Hypersensitivity to Hsp90 inhibitor at 37°C is not a general phenomenon; amphotericin B MIC50 values were 0.27 μg/ml and 0.52 μg/ml at 25°C and 37°C respectively.

    “MIC50 values for AmB was 0.27μg/ml and 0.52 μg/ml at 25°C and 37°C respectively indicating that hypersensitivity to Hsp90 inhibitor observed at 37°C is not a general phenomenon.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Growth at physiological temperature critically depends on Hsp90 function; mild compromise at 37°C but not 25°C is lethal for C. neoformans.

    “We find that growth at physiological temperature critically depends on Hsp90 function as mild compromise in function at 37°C but not at 25°C is lethal for C. neoformans.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hsp90 inhibition is fungicidal at 37°C; C. neoformans grown at 37°C for 72 h in the presence of radicicol were inviable after transfer to 37°C and 25°C.

    “We found that C. neoformans grown at 37°C for 72 hr in the presence of RAD were inviable after transferring to 37°C as well as 25°C indicating that the Hsp90 inhibition is fungicidal (Fig 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    17-AAG inhibits CnHsp90 ATPase activity with IC50 of 117.15 μM at 25°C and 26.89 μM at 37°C.

    “IC50 for 17-AAG-mediated inhibition of CnHsp90 ATPase activity was observed to be 117.15 μM and 26.89 μM at 25°C and 37°C respectively (Fig 3C).”
    Quote in the paperSubstantial claim

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

    Findingown data

    A C. neoformans clinical isolate shows 12.4-fold hypersensitivity to Hsp90 inhibition at 37°C; MIC50 for radicicol was 18.63 μM at 25°C and 1.51 μM at 37°C.

    “A more pronounced effect was seen for a Cryptococcus clinical isolate wherein MIC 50 value was found to be 18.63 μM and 1.51 μM at 25°C and 37°C respectively (Fig 1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hsp90 is enriched in the cell wall, as shown by high co-localization between Hsp90 and calcofluor staining.

    “Also, a high degree of co-localization between Hsp90 and calcofluor staining was observed implicating that Hsp90 is also enriched in the cell wall (Fig 4A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    C. neoformans growth is hypersensitive to Hsp90 inhibition at 37°C relative to 25°C; MIC50 for radicicol was 15.67 μM at 25°C and 1.78 μM at 37°C.

    “MIC50 (minimum inhibitory concentration) for RAD treatment was observed to be 15.67 μM when cells were grown at 25°C. However, at 37°C, there was almost a tenfold reduction in MIC50 (Fig 1A). At 37°C, MIC50 was found to be 1.78 μM.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In Candida albicans, Hsp90 governs yeast-to-hyphal transition, drug resistance, and biofilm formation, and is implicated in pathogenicity.

    “Hsp90, an essential molecular chaperone [1,3,5,6], has been shown to govern various aspects of C. albicans pathogenicity determinants including morphological yeast to hyphal transition [7], emergence of drug resistance [8–10] and biofilm formation [11].”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [7] RS Shapiro, 2009, Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling, Curr Biol source

      Not checked in the free run.

    • cites [8] LE Cowen, 2005, Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi, Science source

      Not checked in the free run.

    • cites [9] LE Cowen, 2006, Genetic architecture of Hsp90-dependent drug resistance, Eukaryot Cell source

      Not checked in the free run.

    • cites [10] SD Singh, 2009, Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin, PLoS Pathog source

      Not checked in the free run.

    • cites [11] N Robbins, 2011, Hsp90 governs dispersion and drug resistance of fungal biofilms, PLoS Pathog source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Thermotolerance is an essential attribute for pathogenesis of Cryptococcus; only two species in the genus that can grow at 37°C are human pathogens.

    “Only two are human pathogens by virtue of their abilities to grow at 37°C implicating the importance of thermotolerance for pathogenesis [21,22].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [21] JR Perfect, 2006, Cryptococcus neoformans: the yeast that likes it hot, FEMS Yeast Res source

      Not checked in the free run.

    • cites [22] BR Steen, 2002, Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans, Genome Res source

      Not checked in the free run.

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

    Study design and conductnone given

    Forty cells were measured for each experiment on at least three separate occasions.

    “40 cells were measured for each experiment on at least three separate occasions.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For cell surface association studies, permeabilization was not carried out.

    “For cell surface association studies, it should be noted that permeabilization step was not carried out.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Targeting Hsp90 can abrogate thermotolerance and hence growth of the fungus.

    “Firstly, it is involved in mediating thermotolerance which implies targeting Hsp90 can abrogate thermotolerance and hence growth of the fungus.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In C. albicans, a 47 kDa C-terminal fragment of Hsp90 is present on the cell wall, and antibodies targeting exposed Hsp90 inhibit infection.

    “Interestingly, in C. albicans, a 47kDa C terminal fragment of Hsp90 was shown to be present on the cell wall [13,14] and antibodies targeting the exposed Hsp90 were shown to inhibit infection [15].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [13] ET Burt, 2003, Isolation and partial characterization of Hsp90 from Candida albicans, Ann Clin Lab Sci

      Not checked in the free run.

    • cites [14] R Matthews, 1988, Characterisation and cellular localisation of the immunodominant 47-Kda antigen of Candida albicans, J Med Microbiol source

      Not checked in the free run.

    • cites [15] F Bugli, 2013, Human monoclonal antibody-based therapy in the treatment of invasive candidiasis, Clin Dev Immunol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Hsp90 is involved in thermotolerance, cell wall integrity, and capsulation processes in C. neoformans, which are essential virulence determinants.

    “In all, our study establishes the involvement of Hsp90 in thermotolerance, cell wall integrity and capsulation processes in C. neoformans which are the most essential virulence determinants of the pathogenic fungus.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Cell viability was examined by Trypan blue dye exclusion to rule out cell death due to radicicol treatment.

    “Cell viability was examined by Trypan blue dye exclusion method to rule out cell death due to RAD treatment.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Results were reported as mean ± S.E.M.; grouped data were analyzed using one-way ANOVA and paired comparisons used a two-tailed P-test.

    “All results were reported as Mean ± S.E.M. Grouped data was statistically analyzed using one-way ANOVA. Two-tailed P-test was used for paired comparisons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Cell viability was 90-95% during RAD treatment, ruling out cell death as the cause of loss of surface Hsp90.

    “We examined the cell viability by Trypan blue dye exclusion method and found 90–95% viability thus ruling out cell death during RAD treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Binding of anti-CnHsp90 antibody to intact yeast cells is specific, as no binding was observed with anti-PGK antibody in control cells.

    “Antibody to cytosolic protein PGK and pre-immune sera served as negative controls for the experiments.”
    • Also stated in: § Hsp90 is localised to the surface of C. neoformans · plain
      “Binding was not observed in control fungal cells using polyclonal antiserum against PGK.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Negative controls ruled out nonspecific antibody binding and accidental permeabilization: anti-PGK antibody did not give cell surface signal, and pre-immune serum did not give cell wall signal.

    “Immunofluorescence using Anti-PGK Ab was performed as above and signal corresponding to cell surface was not observed in this case. Signal at the cell wall was also not seen in case of cells probed with pre-immune serum thereby ruling out nonspecific antibody binding.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    To rule out nonspecific ATPase activity due to copurifying proteins, 300 μM 17-AAG was used as a control.

    “To rule out nonspecific ATPase activity due to copurifying proteins, 300 μM of Hsp90 inhibitor 17-AAG was used as control.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    In Cryptococcus, Hsp90 is crucial for growth and radicicol has synergistic action with azoles.

    “In Cryptococcus, Hsp90 has been recently shown to be crucial for growth of the fungi and Hsp90 inhibitor radicicol has synergistic action with azoles [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] A Cordeiro Rde, 2016, Inhibition of heat-shock protein 90 enhances the susceptibility to antifungals and reduces the virulence of Cryptococcus neoformans/Cryptococcus gattii species complex, Microbiology source

      Not checked in the free run.

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

    Interpretationreasoning

    Higher in vitro catalytic efficiency indicates that the rate of client cycling through the chaperone cycle will be higher, but in vivo it may be regulated by cochaperones and cofactors.

    “Higher in vitro catalytic efficiency indicates that the rate of client cycling through the chaperone cycle will be higher, however, in vivo it may also be regulated by cochaperones and cofactors.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Hsp90 acts as a thermosensor in the malaria parasite and regulates encystation in Entamoeba and Giardia.

    “For instance, Hsp90 acts as a thermosensor in the malaria parasite wherein temperature stress is perceived as a cue for transition from one development stage to another [1]. In Entamoeba and Giardia, Hsp90 regulates the process of encystation [2,3].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [1] G Banumathy, 2003, Heat shock protein 90 function is essential for Plasmodium falciparum growth in human erythrocytes, J Biol Chem source

      Not checked in the free run.

    • cites [2] M Singh, 2015, Heat Shock Protein 90 regulates encystation in Entamoeba, Front Microbiol source

      Not checked in the free run.

    • cites [3] RK Nageshan, 2014, Trans-spliced heat shock protein 90 modulates encystation in Giardia lamblia, PLoS Negl Trop Dis source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Hsp90 trafficking to the cell wall and capsular assembly are related processes, assigning Hsp90 an important role in regulating capsule dynamics in C. neoformans.

    “Taken together, these observations clearly suggest that Hsp90 trafficking to the cell wall and capsular assembly are related processes and hence it assigns an important role of regulating capsule dynamics to Hsp90 in C. neoformans.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.

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

    Premisecitation

    In Aspergillus fumigatus, Hsp90 is involved in drug resistance, conidiation, and maintenance of cell wall integrity.

    “In Aspergillus fumigatus, the role of Hsp90 in drug resistance [16], conidiation and maintenance of cell wall integrity has been well established [17].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] LE Cowen, 2009, Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [17] F Lamoth, 2012, Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus, Eukaryot Cell source

      Not checked in the free run.

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

    Premisereasoning

    High body temperature in mammals and birds provides an innate physical barrier to fungal spores, and the ability to survive at 37°C is the utmost requirement for human pathogenicity.

    “Nonetheless upon summarizing the lessons learned from pathogenic and non-pathogenic fungi, we arrive at the conclusion that high body temperature in mammals and birds provide an innate physical barrier to the vast inoculum of ubiquitous fungal spores. Therefore, the ability to successfully survive at the physiological temperature of 37°C seems to be the utmost requirement in order to be pathogenic”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Hsp90 regulates the yeast-to-hyphal switch in C. albicans by repressing the Ras1-PKA signaling pathway.

    “Interestingly Hsp90 has been shown to be the regulator of this switch by repressing the Ras1-PKA signaling pathway [7].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] RS Shapiro, 2009, Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling, Curr Biol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Presence of Hsp90 on the cell wall indicates that it chaperones important clients involved in capsule processes, and compromising Hsp90 function compromises cell wall organisation which in turn affects capsule assembly.

    “In this light, presence of Hsp90 on the cell wall indicates that it chaperones important clients which are involved in the above processes, hence, compromising Hsp90 function compromises cell wall organisation which in turn affects capsule assembly.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.

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

    Premisecitation

    Cryptococcus species capable of growth at 37°C are human pathogens, while species lacking this ability are non-pathogenic.

    “One such example of thermotolerance manoeuvring fungal pathogenesis has been seen in the genus Cryptococcus wherein only species capable of growth at 37°C are human pathogens. Cryptococcus species which lack this ability despite having other virulence factors, for example, C. podzolicus are non-pathogenic [39].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] S Araujo Gde, 2012, Capsules from pathogenic and non-pathogenic Cryptococcus spp. manifest significant differences in structure and ability to protect against phagocytic cells, PLoS One source

      Not checked in the free run.

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

    Premisecitation

    In Candida albicans and Aspergillus fumigatus, Hsp90 has an established role in echinocandin resistance.

    “In Candida albicans and Aspergillus fumigatus, role of Hsp90 in echinocandin resistance has been well established [10].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] SD Singh, 2009, Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin, PLoS Pathog source

      Not checked in the free run.

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

    Premisecitation

    Previous studies in Cryptococcus have shown synergy of Hsp90 inhibitors Mycograb and RAD with azoles.

    “Previous studies in Cryptococcus have shown synergy of Hsp90 inhibitors Mycograb [15] and RAD [18] with azoles.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [15] F Bugli, 2013, Human monoclonal antibody-based therapy in the treatment of invasive candidiasis, Clin Dev Immunol source

      Not checked in the free run.

    • cites [18] A Cordeiro Rde, 2016, Inhibition of heat-shock protein 90 enhances the susceptibility to antifungals and reduces the virulence of Cryptococcus neoformans/Cryptococcus gattii species complex, Microbiology source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Hsp90 plays a critical role in mediating three essential virulence determinants in C. neoformans: thermotolerance, capsule formation, and echinocandin resistance.

    “Taken together, our study establishes a critical role of Hsp90 in mediating three essential virulence determinants in C. neoformans i.e. thermotolerance, capsule formation and echinocandin resistance.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.

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

    Premisecitation

    Colloidal gold labelled mAbs against GXM bind with linear fibres present both on cell surface and cytosolic secretory vesicles.

    “Interestingly, it was also shown that colloidal gold labelled mAbs against GXM binds with linear fibres present both on cell surface and cytosolic secretory vesicles [38].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] TL Doering, 2009, How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans, Annu Rev Microbiol source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Calcineurin is a bona fide client of Hsp90 and has been implicated in growth at high temperature and virulence of Cryptococcus.

    “For example, calcineurin, a bona fide client of Hsp90, has been implicated in growth at high temperature as well as virulence of the pathogen [47–49].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [47] YL Chen, 2013, Calcineurin governs thermotolerance and virulence of Cryptococcus gattii, G3 (Bethesda) source

      Not checked in the free run.

    • cites [48] DS Fox, 2001, Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans, Mol Microbiol source

      Not checked in the free run.

    • cites [49] A Odom, 1997, Calcineurin is required for virulence of Cryptococcus neoformans, EMBO J source

      Not checked in the free run.

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

    Premisecitation

    The classical secretion pathway has been implicated in capsular monomer assembly using conditionally defective secretory mutants.

    “Role of classical secretion pathway has been implicated in capsular monomer assembly by using conditionally defective secretory mutants wherein cargo from accumulated vesicles was found to react with anti GXM mAbs [37].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] A Yoneda, 2006, A eukaryotic capsular polysaccharide is synthesized intracellularly and secreted via exocytosis, Mol Biol Cell source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In C. neoformans, Hsp90 is upregulated at 37°C, under reduced iron conditions, and under fluconazole stress.

    “In C. neoformans, serial analysis of gene expression and microarray studies have revealed the upregulation of Hsp90 at 37°C [22,40], under reduced iron conditions [41] and under fluconazole stress [42].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [22] BR Steen, 2002, Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans, Genome Res source

      Not checked in the free run.

    • cites [40] PR Kraus, 2004, Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray, Eukaryot Cell source

      Not checked in the free run.

    • cites [41] T Lian, 2005, Iron-regulated transcription and capsule formation in the fungal pathogen Cryptococcus neoformans, Mol Microbiol source

      Not checked in the free run.

    • cites [42] AR Florio, 2011, Genome-wide expression profiling of the response to short-term exposure to fluconazole in Cryptococcus neoformans serotype A, BMC Microbiol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Hsp90 is involved both in assembly and maintenance of capsule.

    “Upon compromise of Hsp90 function during the process of capsulation as well as post capsulation, significant reduction in capsule size was seen thereby confirming that Hsp90 is involved both in assembly and maintenance of capsule.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.
    • Rests on an assumption: The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.

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

    Premisecitation

    Kinases Hog1, Mkc1 and Cek1 are crucial for cell wall integrity and are well-established clients of Hsp90.

    “Kinases like Hog1, Mkc1 and Cek1, which are crucial for cell wall integrity [50], are also well- established clients of Hsp90 [12,51].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [50] B Eisman, 2006, The Cek1 and Hog1 mitogen-activated protein kinases play complementary roles in cell wall biogenesis and chlamydospore formation in the fungal pathogen Candida albicans, Eukaryot Cell source

      Not checked in the free run.

    • cites [12] MD Leach, 2012, Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast, PLoS Pathog source

      Not checked in the free run.

    • cites [51] S Diezmann, 2012, Mapping the Hsp90 genetic interaction network in Candida albicans reveals environmental contingency and rewired circuitry, PLoS Genet source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The combination effect of RAD and anidulafungin is seen at 37°C and not at 25°C, reconfirming that virulence and thermotolerance are linked in this fungus.

    “Interestingly this effect is also seen at 37°C and not at 25°C, thus again reconfirming the fact that virulence and thermotolerance are linked in this fungus.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.
44 of 44

  • The paper relies on this without support.

    The mechanism of growth at elevated temperature with respect to heat shock response has long been enigmatic.

    “However, the mechanism of growth at elevated temperature with respect to heat shock response has long been enigmatic.”

  • The paper relies on this without support.

    Biochemical parameters of Hsp90 from fungal pathogens have not been characterized previously.

    “The biochemical parameters of Hsp90 from fungal pathogens have not been characterized previously.”

  • The paper relies on this without support.

    Hsp90s from different organisms differ in their biochemical activities.

    “However, it is a well-established fact that Hsp90s from different organisms differ in their biochemical activities.”

  • The paper relies on this without support.

    RAD specifically inhibits Hsp90 function in C. neoformans under the conditions used.

    “we also grew the cells in the presence of 0.5 μMRAD for 48 hours and probed Hsp90 surface localisation as above.”
    “Compromising Hsp90 function with RAD (0.25μM and 0.5 μM) in presence of AF (4μg/ml)) had no impact on growth of the isolate as compared to the drug free controls at 25°C.”

  • The paper relies on this without support.

    BFA specifically blocks ER-to-Golgi anterograde transport in C. neoformans under the conditions used.

    “BFA interferes with the action of ARF and blocks the anterograde transport of proteins between the ER and Golgi apparatus.”

  • The paper relies on this without support.

    NEM specifically interferes with disulphide bond formation in C. neoformans under the conditions used.

    “N-ethylmaleimide (NEM) which is a cysteine alkylating agent that interferes with disulphide bond formation.”

  • The paper relies on this without support.

    The capsule-inducing conditions used (10% serum, 37°C with 5% CO2) mimic the host environment and induce capsulation.

    “we tried to mimic the host environment by growing cells in the presence of 10% serum, which also is known to induce the process of capsulation.”
    “another well-established capsule inducing condition i.e. 37°C and 5% CO2 which mimics lung environment”
7 of 7

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?

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.

  • Abstract reverses the effect of Hsp90 inhibition on anidulafungin tolerance

    Major

    The abstract says compromising Hsp90 improves anidulafungin tolerance, whereas the results say it reduces tolerance and decreases growth under combination treatment.

  • Cloning source is described as both cDNA and genomic DNA

    Minor

    The methods say CnHsp90 was amplified from cDNA for cloning into pRSET-A, whereas the results say it was cloned from genomic DNA into that vector.

  • Figure 2 caption mislabels the temperature of the 6.39 × 10−5 value

    Minor

    The caption labels both catalytic-efficiency values as measured at 25°C, despite saying they correspond to 25°C and 37°C, respectively. The results identify 6.39 × 10−5 as the 37°C value.

  • Figure 3 caption reverses the temperature-specific IC50 values

    Minor

    The caption assigns 26.89 μM to 25°C and 117.15 μM to 37°C, but the results assign those values to 37°C and 25°C, respectively. The caption's assignment also contradicts its claim that inhibition is more potent at 37°C.

4 of 4

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Authors
Chatterjee, S., & Tatu, U.
Affiliations
Indian Institute of Science Bangalore (IN)
Corresponding author
  • Chatterjee, Sharanya
  • Tatu, Utpal
ORCID
Venue
PLOS Neglected Tropical Diseases
Publisher
Public Library of Science (PLoS)
Published
2017-08-04
Volume
11
Issue
8
Pages
e0005836
ISSN
  • 1935-2727 (ISSN-L)
  • 1935-2735
DOI
10.1371/journal.pntd.0005836
PMC
PMC5559104
OpenAlex
W2742305482
Semantic Scholar
105b17274d094dddaf62dd902cfee336c31b8809
Cited by
57
References
52
Citations by year
2026: 6 · 2025: 8 · 2024: 4 · 2023: 8 · 2022: 9 · 2021: 9 · 2020: 5 · 2019: 2 · 2018: 5 · 2017: 1
FWCI
2.79
Type
journal article
Language
en
Update notices
new_version (2017-08-16)
Open access
Licence
Locations
  • PLoS neglected tropical diseases (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • NOT FOUND REPOSITORY (Indian Institute of Science Bangalore) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Department of Biotechnology, Ministry of Science and Technology
  • Department of Science and Technology, Ministry of Science and Technology (DST INSPIRE)
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
HSP90, Cryptococcus neoformans, heat tolerance, fungal virulence, cell surface localization, capsule formation, melanization, 17-AAG, Hsp90 ATPase activity, antifungal resistance, fungal pathogenesis
MeSH terms
Thermotolerance, Anidulafungin, Cryptococcosis, Cryptococcus neoformans, Fungal Proteins, Humans, Microbial Sensitivity Tests, Temperature, Virulence, Benzoquinones, HSP90 Heat-Shock Proteins, Virulence Factors, Lactams, Macrocyclic, Echinocandins

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.

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