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Cited by 114Fungal and yeast genetics researchAntifungal resistance and susceptibilityFungal Plant Pathogen Control

Abnormal Ergosterol Biosynthesis Activates Transcriptional Responses to Antifungal Azoles

Open access
Hu, Chengcheng, Zhou, Mi, Wang, Wenzhao, Sun, Xianyun, Yarden, Oded, Li, Shaojie·2018·Frontiers in Microbiology
DOI 10.3389/fmicb.2018.00009PMC PMC5776110
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Abstract

Fungi transcriptionally upregulate expression of azole efflux pumps and ergosterol biosynthesis pathway genes when exposed to antifungal agents that target ergosterol biosynthesis. To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol. Using an inducible promoter to regulate Neurospora crassa erg11, which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles. By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by cdr4 and, unexpectedly, genes encoding ergosterol biosynthesis enzymes (erg genes) that are responsive to azoles, are not dependent on ergosterol depletion. Nonetheless, deletion of erg2, which encodes C-8 sterol isomerase, also induced expression of cdr4. Deletion of erg2 also induced the expression of erg24, the gene encoding C-14 sterol reductase, but not other tested erg genes which were responsive to erg11 inactivation. This indicates that inhibition of specific steps of ergosterol biosynthesis can result in different transcriptional responses, which is further supported by our results obtained using different ergosterol biosynthesis inhibitors. Together with the sterol profiles, these results suggest that the transcriptional responses by cdr4 and erg genes are associated with accumulation of specific sterol intermediate(s). This was further supported by the fact that when the erg2 mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway. Even though cdr4 expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of cdr4 did not result in efflux of the accumulated intermediate(s). This study demonstrates, by detailed genetic and chemical analysis, that transcriptional responses by a major efflux pump and genes of the ergosterol biosynthesis pathway to ergosterol biosynthesis inhibitors can be independent of the presence of the drugs and are linked with the accumulation of ergosterol intermediate(s).

AI snapshot

Written from the paper’s full text.

TL;DR

In *Neurospora crassa*, transcriptional responses of the azole efflux pump gene *cdr4* and several ergosterol biosynthesis genes can be activated by defects in ergosterol biosynthesis even without azole drugs. These responses are linked to accumulation of specific sterol intermediates rather than ergosterol depletion.

Key findings

  • In a copper-regulated *Ptcu-1::erg11* strain, inactivation of *erg11* without ketoconazole induced *cdr4*, *erg5*, *erg24*, and *erg6* by at least 28-, 5-, 6.5-, and 33-fold, respectively, similar to ketoconazole treatment.
  • Ergosterol depletion alone was not sufficient: Δ*erg3*, Δ*erg4*, and Δ*erg5* mutants showed no changes in tested azole-responsive genes, whereas Δ*erg2* increased *cdr4* and *erg24* expression by 7- and 10-fold, respectively.
  • Different ergosterol biosynthesis inhibitors caused different transcriptional responses: ketoconazole increased *cdr4* about 13-fold, amorolfine about 5-fold, while terbinafine had no effect on *cdr4*; amorolfine induced *erg24* 6-fold and *erg2* 4-fold.
  • In Δ*cdr4*, ketoconazole accumulation was 2.4-fold higher than wild type, and ketoconazole-treated Δ*cdr4* showed about a threefold greater reduction in total ergosterol and increased lanosterol, eburicol, and 14α-methyl-3,6-diol at 1.6-, 1.7-, and 2.8-fold of wild type, respectively; no free ergosterol, lanosterol, eburicol, or 14α-methyl-3,6-diol was detected extracellularly.

Methods

  • Used *N. crassa* wild type, parental *bd ku70RIP*, a copper-responsive *Ptcu-1::erg11* strain, complemented *Perg11::erg11*, and deletion mutants Δ*erg2*, Δ*erg3*, Δ*erg4*, Δ*erg5*, and Δ*cdr4*.
  • Treated cultures with ketoconazole (2 mg/L), amorolfine (0.375 mg/L), or terbinafine (3 mg/L), and analyzed gene expression by qRT-PCR normalized to β-tubulin using the 2-ΔΔCt method, with three biological replicates.
  • Measured intracellular and extracellular sterols and ketoconazole by HPLC-MS, and detected tagged ERG11 by Western blotting with anti-myc antibodies.

Limitations

  • The paper states that further genome-wide transcriptional profiling is needed for a comprehensive view of responses to ergosterol biosynthesis inhibitors, and that how different sterol intermediates activate transcriptional responses requires further elucidation.
  • The paper also states that why and how efflux pumps are activated by sterol intermediates remains to be elucidated.
  • Evident from the text: the study is limited to *N. crassa*, and the specific regulatory factors linking sterol intermediate accumulation to *cdr4* and *erg* gene transcription are not identified.

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

Integrity signals

Registry

Crossref · 10.3389/fmicb.2018.00009

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 49 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Transcriptional induction of cdr4 did not result in efflux of accumulated sterol intermediate(s), based on intra- and extracellular sterol analysis by HPLC-MS.

    “Even though cdr4 expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of cdr4 did not result in efflux of the accumulated intermediate(s).”
    Quote in the paperSubstantial claim
  • Findingown data

    In the erg2 mutant treated with ketoconazole, upstream inhibition overrode the effects of downstream inhibition on the ergosterol biosynthesis pathway.

    “This was further supported by the fact that when the erg2 mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway.”
    Quote in the paperSubstantial claim
  • Findingown data

    In Neurospora crassa, the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles.

    “Using an inducible promoter to regulate Neurospora crassa erg11, which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles.”
    • Also stated in: § Inactivation of erg11 Causes Transcriptional Responses Similar to Those Following Ketoconazole Treatment · plain
      “Transcript levels of cdr4 as well as those of ergosterol biosynthesis genes were all significantly higher in the Ptcu-1::erg11 strain, in spite of the lack of KTC in the medium.”
    Quote in the paperSubstantial claim
  • Findingown data

    Deletion of erg2 in N. crassa induces expression of cdr4.

    “Nonetheless, deletion of erg2, which encodes C-8 sterol isomerase, also induced expression of cdr4.”
    • Also stated in: § Ergosterol Depletion Is Not the Cause for Activating Stress Response · plain
      “Interestingly, in a Δerg2 background, expression of cdr4 and erg24 were increased by 7 and 10-folds, respectively, while other genes tested were not affected (Figure 4D).”
    Quote in the paperSubstantial claim
  • Findingown data

    Transcriptional responses of cdr4 and azole-responsive ergosterol biosynthesis genes in N. crassa are not dependent on ergosterol depletion.

    “By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by cdr4 and, unexpectedly, genes encoding ergosterol biosynthesis enzymes (erg genes) that are responsive to azoles, are not dependent on ergosterol depletion.”
    • Also stated in: § Ergosterol Depletion Is Not the Cause for Activating Stress Response · plain
      “Thus, the stress responses to azoles by cdr4 and ergosterol biosynthesis genes are not activated by ergosterol depletion.”
    Quote in the paperSubstantial claim
  • May combine two claims. 36
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Findingown data

    Deletion of erg2 in N. crassa induces expression of erg24 but not other tested erg genes responsive to erg11 inactivation.

    “Deletion of erg2 also induced the expression of erg24, the gene encoding C-14 sterol reductase, but not other tested erg genes which were responsive to erg11 inactivation.”
    • Also stated in: § Ergosterol Depletion Is Not the Cause for Activating Stress Response · plain
      “Interestingly, in a Δerg2 background, expression of cdr4 and erg24 were increased by 7 and 10-folds, respectively, while other genes tested were not affected (Figure 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In N. crassa, erg11 is required for hyphal elongation but not for germination.

    “This result indicates that erg11 is required for hyphal elongation but not germination.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In Ptcu-1::erg11 shifted to CuSO4, erg11 expression was twofold lower than the parental strain and ERG11 protein was undetectable.

    “Its erg11 expression was twofold lower than the parental strain and the protein was undetectable (Figure 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In N. crassa wild type, amorolfine and ketoconazole treatments increased cdr4 expression by almost 5- and 13-fold, respectively.

    “AMOR or KTC treatments led to an almost 5- and 13-fold increase in cdr4 expression, respectively (Figure 5), verifying that sterol inhibition does, in fact, induce cdr4 transcription.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AMOR treatment only induced erg24 by 6 folds but not other erg genes in N. crassa.

    “AMOR treatment only induced erg24 by 6 folds but not other erg genes (Figure 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In N. crassa, Δerg4 grows approximately 80% of wild type with compact and fluffy colony morphology.

    “Deletion of erg4 also resulted in slow hyphal growth, albeit in a less severe manner than Δerg2, as this strain grew approximately 80% of wild type, yet its colony morphology was more compact and fluffy in appearance (Figure 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    N. crassa deletion mutants Δerg2, Δerg3, Δerg4 and Δerg5 are viable.

    “All the mentioned gene deletion mutants are viable (Figure 4A), similarly to that described in S. cerevisiae (Lees et al., 1995).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Lees et al., 1995 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In N. crassa, Δerg4 accumulates ergosta-5,7,22,24(28)-tetraenol; Δerg5 accumulates ergosta-5,7,24(28)-trienol and ergosta-5,7-dienol; Δerg2 accumulates ergosta-5,8,22-trienol and fecosterol.

    “Rather, ergosta-5,7,22,24(28)-tetraenol was accumulated in the Δerg4 strain and ergosta-5,7,24(28)-trienol and ergosta-5,7-dienol were accumulated in the Δerg5 strain when compared to wild type (Figure 4B and Supplementary Table 4). In the Δerg2 strain, ergosta-5,8,22-trienol and fecosterol were accumulated (Supplementary Table 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Ptcu-1::erg11 treated with CuSO4, ergosterol was reduced to approximately 56% of BCS-treated levels and lanosterol, eburicol and 14α-methyl-3,6-diol accumulated, similar to azole-treated N. crassa.

    “CuSO4 treatment resulted in the reduction of ergosterol to a level of approximately 56% of that treated with BCS along with the accumulation of lanosterol, eburicol and 14α-methyl-3,6-diol, similar to that found in N. crassa treated with azoles (Chen et al., 2016).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Chen et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In N. crassa Δerg2, expression of cdr4 and erg24 increased by 7- and 10-fold, respectively, while other tested genes were not affected.

    “Interestingly, in a Δerg2 background, expression of cdr4 and erg24 were increased by 7 and 10-folds, respectively, while other genes tested were not affected (Figure 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TERB treatment resulted in accumulation of squalene and an unknown compound.

    “TERB treatment resulted in accumulation of squalene and an unknown compound”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In Ptcu-1::erg11 with erg11 inactivation, mRNA levels of cdr4, erg5, erg24 and erg6 were induced by at least 28-, 5-, 6.5- and 33-fold, respectively, similar to KTC treatment.

    “the mRNA levels of cdr4, erg5 (NCU05278, encoding sterol C-22 desaturase), erg24 (NCU08762, encoding sterol C-14 reductase) and erg6 (NCU03006, encoding sterol C-24 methyltransferase) were dramatically induced, by at least 28, 5, 6.5, and 33-fold, respectively, in a manner similar to the levels of induction imposed by the KTC treatment (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Disruption at different steps in ergosterol biosynthesis had different effects and resulted in difference in sterol profiles.

    “Our results also showed that disruption at different steps in ergosterol biosynthesis had different effects and resulted in difference in sterol profiles.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Genetic disruption of erg4 and erg5 in N. crassa did not cause transcriptional response by any of the tested genes in ergosterol biosynthesis, although ergosterol formation was completely abolished.

    “Surprisingly, we found that genetic disruption of erg4 and erg5 in N. crassa did not cause transcriptional response by any of the tested genes in ergosterol biosynthesis, although ergosterol formation was completely abolished.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of erg5, erg6 and erg11 in the Δerg2 mutant treated with KTC showed a similar response to KTC induction on its own, which is very different from that in the non-treated Δerg2 strain.

    “The expression of erg5, erg6 and erg11 in the Δerg2 mutant treated with KTC also showed a similar response to KTC induction on its own, which is very different from that in the non-treated Δerg2 strain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Accumulation of specific sterols but not ergosterol depletion was associated with the transcriptional response by cdr4.

    “Our results showed that accumulation of specific sterols but not ergosterol depletion was associated with the transcriptional response by cdr4 (Figure 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In wild type, KTC treatment resulted in a reduction of total ergosterol content and abnormal accumulation of sterols along the biosynthetic pathway.

    “In wild type, KTC treatment resulted in a reduction of total ergosterol content and the abnormal accumulation of sterols along the biosynthetic pathway (Figure 8B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of erg24 in the Δerg2 mutant treated with KTC was reduced compared to the Δerg2 mutant (nine folds) and similar to that observed in wild type treated with the drug.

    “The expression of erg24 in the Δerg2 mutant treated with KTC was reduced when compared to that in the Δerg2 mutant (nine folds) and similar to that observed in wild type treated with the drug.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    KTC treatment of the Δerg2 strain led to accumulation of lanosterol, eburicol and 14α-methyl-3,6-diol along with depletion of ergosta-5,8,22-trienol.

    “KTC treatment of this strain also lead to accumulation of lanosterol, eburicol and 14α-methyl-3,6-diol along with the depletion of ergosta-5,8, 22-trienol”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of cdr4 was induced 3 h after KTC treatment while erg11 expression was induced within 1 h in the presence of the drug.

    “the expression of cdr4 was induced 3 h after KTC treatment while erg11 expression was induced within 1 h in the presence of the drug (Figure 7).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Ergosterol homeostasis is tightly maintained by several important regulators and is imperative for ergosterol biosynthesis and cell membrane functions.

    “Ergosterol homeostasis, which is tightly maintained by several important regulators, is imperative for ergosterol biosynthesis and cell membrane functions (Bien and Espenshade, 2010; Maguire et al., 2014).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bien and Espenshade, 2010 source

      Not checked in the free run.

    • cites Maguire et al., 2014 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    These transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol.

    “To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Thakur et al., 2008 source

      Not checked in the free run.

    • cites Hughes et al., 2005 source

      Not checked in the free run.

    • cites Yang et al., 2015 source

      Not checked in the free run.

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

    Premisecitation

    The efflux pump system is present in almost all species and plays important roles in drug resistance in bacteria, fungi and human cancer cells by efflux of the drugs.

    “The efflux pump system, which is comprised of pumps located in the cell membrane and their regulators, is present in almost all species and plays an important roles in drug resistance in bacteria, fungi and human cancer cells by efflux of the drugs (Golin et al., 2007; Blair et al., 2014; Cowen et al., 2014; Paul and Moye-Rowley, 2014; Tanwar et al., 2014; Kathawala et al., 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Golin et al., 2007 source

      Not checked in the free run.

    • cites Blair et al., 2014 source

      Not checked in the free run.

    • cites Cowen et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In S. cerevisiae, transcription factor Upc2p regulates expression of ergosterol biosynthesis genes, directly interacts with ergosterol, and its activity is regulated by ergosterol levels.

    “In S. cerevisiae, transcription factor Upc2p regulates expression of ergosterol biosynthesis genes. Upc2p directly interacts with ergosterol and its activity was regulated by ergosterol levels (Yang et al., 2015).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Yang et al., 2015 source

      Not checked in the free run.

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

    Premisecitation

    In S. cerevisiae, transcription factors Pdr1p and Pdr3p can be activated by directly interacting with xenobiotics, including ketoconazole, and thus might act as sensors of xenobiotics.

    “In S. cerevisiae, the transcription factors Pdr1p and Pdr3p, which regulate expression of efflux pumps, can be activated by directly interacting with xenobiotics, including ketoconazole, and thus might act as sensors of xenobiotics (Thakur et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Thakur et al., 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    The accumulation of sterol intermediates is also possible to induce the transcriptional responses.

    “Thus, the accumulation of sterol intermediates is also possible to induce the transcriptional responses.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Upon azole stress, fungi can respond with a change in the transcription of a variety of genes.

    “Upon azole stress, fungi can respond with a change in the transcription of a variety of genes.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Agarwal et al., 2003 source

      Not checked in the free run.

    • cites Liu et al., 2005, 2010

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

    • cites Sun et al., 2014 source

      Not checked in the free run.

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

    Study design and conductnone given

    Three independent experiments were carried out and each cDNA sample was analyzed at least in duplicate.

    “Three independent experiments were carried out and each cDNA sample was analyzed at least in duplicate.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The azole drug itself was shown to activate gene expression of azole efflux pumps.

    “For transcriptional responses of azole efflux pumps, the azole drug itself was shown to activate gene expression of azole efflux pumps.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Thakur et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    CDR4 may not be involved in expulsion of terbinafine, or terbinafine inhibition of ergosterol biosynthesis cannot induce cdr4 expression.

    “TERB had no effect on cdr4 expression (Supplementary Figure 2), suggesting that this pump may not be involved in expulsion of the compound from the cell, or, the inhibition of ergosterol biosynthesis by TERB cannot induce the expression of this pump.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Terbinafine targets squalene epoxidase ERG1, upstream of ERG11, while amorolfine targets ERG2 and ERG24 downstream of ERG11.

    “Different from azoles, TERB targets squalene epoxidase ERG1 (NCU08280), an enzyme upstream of sterol 14α-demethylase ERG11, while AMOR targets two enzymes downstream of ERG11 in the ergosterol biosynthetic pathway, sterol C8-C7 isomerase ERG2 and C-14 sterol reductase ERG24 (Lupetti et al., 2002) (Figure 1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Lupetti et al., 2002 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Transcription factors such as Tac1p and Upc2p govern the expression of many azole-responsive genes and confer azole resistance.

    “In addition, transcription factors, such as Tac1p that regulates the azole efflux pumps and Upc2p that regulates ergosterol biosynthesis genes in C. albicans, govern the expression of many azole-responsive genes and confer azole resistance (Coste et al., 2004; Coste et al., 2006; Dunkel et al., 2008; Heilmann et al., 2010; Flowers et al., 2012; Vasicek et al., 2014).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Coste et al., 2004 source

      Not checked in the free run.

    • cites Coste et al., 2006 source

      Not checked in the free run.

    • cites Flowers et al., 2012 source

      Not checked in the free run.

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

    Study design and conductown data

    The results presented are means of three biological replicates.

    “The results presented here are means of three biological replicates, and the significant levels between the two strains in each treatment were calculated by t-test and marked as ∗(p < 0.05), ∗∗(p < 0.01) or ∗∗∗(p < 0.001).”
    • Also stated in: § Ergosterol Depletion Is Not the Cause for Activating Stress Response · plain
      “The results presented here are means of three biological replicates, and the significant levels of cdr4 and erg24 between WT and Δerg2 were calculated by t-test and marked as ∗(p < 0.05), ∗∗(p < 0.01) or ∗∗∗(p < 0.001).”
    • Also stated in: § Differential Responses to Ergosterol Biosynthesis Inhibition in N. crassa · plain
      “The results presented here are means of three biological replicates. The significant levels were calculated by t-test and marked as ∗(p < 0.05), ∗∗(p < 0.01) or ∗∗∗(p < 0.001).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    All above lines of evidence suggest that ergosterol depletion resulted from azole stress might be a cause for the transcriptional responses by ergosterol biosynthesis genes, but some direct experimental data in other fungi are still required for making a definite conclusion.

    “All above lines of evidence suggest that ergosterol depletion resulted from azole stress might be a cause for the transcriptional responses by ergosterol biosynthesis genes, but some direct experimental data in other fungi are still required for making a definite conclusion.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Hughes et al., 2005 source

      Not checked in the free run.

    • cites Porter et al., 2010 source

      Not checked in the free run.

    • cites Yang et al., 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The significance of differences between samples was evaluated by an unpaired two-tailed Student's t-test with p < 0.05 considered significant.

    “The significance of the differences between each two samples were evaluated by an unpaired two-tailed Student’s t-test using GraphPad Prism 7.0 and a p-value of less than 0.05 was considered significant.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In S. pombe, ergosterol depletion activated the expression of ergosterol biosynthesis genes and their regulator Sre1p.

    “In Schizosaccharomyces pombe, it was reported that ergosterol depletion activated the expression of ergosterol biosynthesis genes and their regulator Sre1p (Hughes et al., 2005; Porter et al., 2010).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Hughes et al., 2005 source

      Not checked in the free run.

    • cites Porter et al., 2010 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Azole-caused ergosterol depletion might activate transcriptional response via Upc2p.

    “Thus, azole-caused ergosterol depletion might activate transcriptional response via Upc2p.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In clinical and agricultural azole-resistant isolates, overexpression of sterol 14α-demethylase or azole efflux pumps are among the major causes for azole resistance.

    “In clinical and agricultural azole-resistant isolates, overexpression of sterol 14α-demethylase or azole efflux pumps are among the major causes for azole resistance (White, 1997; Perea et al., 2001; Perea et al., 2002; Redding et al., 2003; Cools et al., 2013; Price et al., 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites White, 1997 source

      Not checked in the free run.

    • cites Perea et al., 2001 source

      Not checked in the free run.

    • cites Cools et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In N. crassa, deletion of erg2 or erg4 causes growth defects, whereas deletion of erg3 or erg5 does not.

    “Thus, deletions in two of the analyzed genes encoding enzymes catalyzing last steps in ergosterol biosynthesis resulted in growth defects, while deletion of the others (erg3 and erg5) did not.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The accumulated toxic 14α-methyl-3,6-diol was thought to cause membrane stresses and activate stress responses with unclear mechanisms.

    “In addition, the accumulated toxic 14α-methyl-3,6-diol was also thought to cause membrane stresses and activate stress responses with unclear mechanisms (Watson et al., 1989; Shapiro et al., 2011).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Watson et al., 1989 source

      Not checked in the free run.

    • cites Shapiro et al., 2011 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Transcriptional responses to disruption at different steps in ergosterol biosynthesis were also different, suggesting a correlation between the sterol profiles and transcriptional responses.

    “Interestingly, the transcriptional responses to disruption at different steps in ergosterol biosynthesis were also different, suggesting a correlation between the sterol profiles and transcriptional responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    This is markedly different from several other fungi, where genes for ergosterol biosynthesis are induced by ergosterol depletion, indicating different regulatory mechanisms among fungi.

    “This is markedly different from those observed in several other fungi, where genes for ergosterol biosynthesis are induced by ergosterol depletion, indicating different regulatory mechanisms among fungi.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Lack or aberrant accumulation of different ergosterol intermediates should cause different functional defects in fungi and induce different transcriptional responses.

    “Thus, lack or aberrant accumulation of different ergosterol intermediates should cause the different functional defects in fungi and induce different transcriptional responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    KTC treatment in the Δerg2 strain could result in depletion of sterol intermediates normally accumulated in the Δerg2 strain and accumulation of sterol intermediates resulting from azole inhibition.

    “Overall, KTC treatment in the Δerg2 strain could result in the depletion of sterol intermediates accumulated normally in the Δerg2 strain and the accumulation of sterol intermediates resulted from azole inhibition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CDR4 is the transporter of azoles, functionally the same as its ortholog Pdr5p in S. cerevisiae.

    “confirming CDR4 is the transporter of azoles, functionally the same to its ortholog Pdr5p in S. cerevisiae.”
    Quote in the paperSubstantial claim
45 of 45

  • The paper relies on this without support.

    The phenotypes and transcriptional changes observed in the Δerg2 mutant are caused by the erg2 deletion and not by secondary mutations.

    “Transformants were screened on plates containing 150 mg/L Hygromycin B and the gene replacement was verified by PCR.”

  • The paper relies on this without support.

    Responses to disruption at other steps in ergosterol biosynthesis were previously unknown.

    “Responses to disruption at other steps in ergosterol biosynthesis were previously unknown.”

  • The paper relies on this without support.

    It is not known which sterol intermediates may be involved in the transcriptional responses.

    “However, it is not known which sterol intermediates may involve in the transcriptional responses.”
3 of 3

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.

  • Conflicting CuSO4 concentrations for the gene-expression experiment

    Minor

    The RNA-preparation method says the cultures were transferred to medium containing 200 μM CuSO4, whereas the Figure 3 caption describes the gene-expression experiment as using 100 μM CuSO4.

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The citation lists haven’t been fetched yet

References 74 · Cited by 114

Research works printed but not linked: 1

  1. Vogel, 1956, A convenient growth medium for Neurospora (medium N)., Microb. Genet. Bull., 13, 42

Authors
Hu, C., Zhou, M., Wang, W., Sun, X., Yarden, O., & Li, S.
Affiliations
  • Chinese Academy of Sciences (CN)
  • Hebrew University of Jerusalem (IL)
Corresponding author
Li, Shaojie
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2018-01-17
Volume
9
Pages
9-9
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2018.00009
PMC
PMC5776110
OpenAlex
W2783348063
Semantic Scholar
825925084764e9b9fb69e37fae088bbbaf877a37
Cited by
114
References
74
Citations by year
2026: 14 · 2025: 11 · 2024: 15 · 2023: 9 · 2022: 9 · 2021: 27 · 2020: 16 · 2019: 11 · 2018: 2
FWCI
4.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (31461143002)
  • Israel Science Foundation
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 › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Fungal Plant Pathogen Control
Keywords
ergosterol biosynthesis, azole antifungals, ERG11, sterol 14α-demethylase, ERG2, Neurospora crassa, transcriptional regulation, sterol intermediates, ketoconazole, ergosterol depletion

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