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Cited by 232Fungal and yeast genetics researchSignaling Pathways in DiseaseAntifungal resistance and susceptibility

CRZ1 , a target of the calcineurin pathway in Candida albicans

Open access
Karababa, Mahir, Valentino, Emilio, Pardini, Giacomo, Coste, Alix T., Bille, Jacques, Sanglard, Dominique·2006·Molecular Microbiology
DOI 10.1111/j.1365-2958.2005.05037.x
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Abstract

Summary: Calcineurin is a major player in calcium‐dependent signal transduction pathways of eukaryotes. Calcineurin acts on transcription factors (e.g. CRZ1 in Saccharomyces cerevisiae ) and governs the expression of genes in a species‐dependent fashion. In Candida albicans , the calcineurin pathway is involved in tolerance to antifungal agents, cation homeostasis and virulence. However, the components of the calcineurin pathway are still poorly investigated in this yeast species. Taking S. cerevisiae as a model to reconstitute this pathway, two CRZ1 ‐like genes, CRZ1 and CRZ2 (for c alcineurin‐ r esponsive z inc finger 1 and 2 genes), were found with C 2 H 2 zinc finger domains. Only CRZ1 was able to restore the calcium hypersusceptibility of a S. cerevisiae crz1 Δ mutant and to mediate calcium‐dependent gene expression in this yeast species. Several experiments showed that CRZ1 was dependent on calcineurin in C. albicans : (i) phenotypic analysis of a crz1 Δ/Δ mutant showed impaired growth as compared with the wild type in the presence of cations (Ca 2+ , Mn 2+ ) as does a mutant lacking calcineurin subunit A ( cna Δ/Δ) and (ii) a green fluorescent protein (GFP)–Crz1p fusion protein showed a calcium‐ and calcineurin‐dependent nuclear localization. To further analyse the relationship between calcineurin and CRZ1 , a comprehensive analysis of calcineurin/Crz1p‐dependent gene expression following addition of Ca 2+ (200 mM) was performed. Among the expression of 264 genes altered by at least twofold, the upregulation of 60 genes was dependent on both calcineurin and CRZ1 . Interestingly, a motif [5′‐G(C/T)GGT‐3′] with similarity to the target sequence of Crz1p (GNGGCG/TCA) from S. cerevisiae was identified as a putative regulatory sequence in the upstream regions of these calcineurin/Crz1p‐dependent genes. However, additional experiments showed that calcineurin may have other targets in addition to CRZ1 . First, CRZ1 was not involved in tolerance to antifungal agents (fluconazole, terbinafine) on the opposite to calcineurin. Second, CRZ1 was only moderately influencing virulence in a mice model of infection which is in sharp contrast to the strong avirulence of cna Δ/Δ mutant in the same animal model. Even though this work establishes CRZ1 as a calcineurin target, further studies are needed to identify other calcineurin‐dependent elements in C. albicans .

AI snapshot

Written from the paper’s full text.

TL;DR

This study identifies *Candida albicans* CRZ1 as a calcineurin-responsive transcription factor and functional homologue of *S. cerevisiae* Crz1p, showing calcium- and calcineurin-dependent nuclear localization and regulation of a large subset of calcium-activated genes. However, CRZ1 does not account for all calcineurin-dependent phenotypes, especially antifungal tolerance and full virulence, indicating additional calcineurin targets.

Key findings

  • Among two *CRZ1*-like genes, only *CRZ1* complemented the calcium hypersensitivity of a *S. cerevisiae crz1Δ* mutant and activated CDRE-dependent reporter expression in a calcium- and FK506-sensitive manner; *CRZ2* did not.
  • A *C. albicans crz1Δ/Δ* mutant showed impaired growth on Ca²⁺ and Mn²⁺ but not Li⁺, was less susceptible than *cnaΔ/Δ*, and unlike *cnaΔ/Δ* retained tolerance to terbinafine; *CRZ2* deletion produced no tested phenotype.
  • GFP-Crz1p localized to the nucleus after calcium exposure and remained cytosolic with FK506; protein A-Crz1p showed calcium- and calcineurin-dependent mobility shifts, consistent with post-translational modification.
  • Microarray analysis of 6039 genes found 264 calcium-regulated genes (≥2-fold), of which 69 were calcineurin-dependent and 65 Crz1p-dependent; 60 genes were regulated by both. A putative CDRE-like motif [5′-G(C/T)GGT-3′] was enriched in their promoters, and mutation of the *UTR2* promoter CDRE-like sequence abolished calcium induction.
  • In mice, wild-type infection killed all mice within 9 days, *cnaΔ/Δ* mice survived 30 days, and *crz1Δ/Δ* infection led to 30% survival at 30 days; kidney burdens at day 3 were approximately 10⁶ cfu g⁻¹ for wild type/revertant, 10² for *cnaΔ/Δ*, and 10⁴ for *crz1Δ/Δ*.

Methods

  • *C. albicans* CRZ1 and CRZ2 were disrupted using the Ura-blaster strategy; complementation and CDRE reporter assays were performed in *S. cerevisiae*.
  • Crz1p localization and modification were assayed with GFP- and protein A-tagged fusions under inducible or constitutive promoters, with calcium and FK506 treatments.
  • Genome-wide expression profiling used *C. albicans* cDNA microarrays covering 6039 genes after 200 mM CaCl₂ exposure at 20, 40, and 60 min in wild-type, *cnaΔ/Δ*, and *crz1Δ/Δ* strains; data were analyzed with ANOVA, twofold thresholds, and GeneSpring clustering.
  • Virulence was tested in a BALB/c mouse intravenous infection model with groups of 10 mice, survival monitoring for 30 days, and tissue burden counts in kidneys and spleen.

Limitations

  • The paper states that further studies are needed to identify other calcineurin-dependent elements in *C. albicans*, because CRZ1 did not explain calcineurin-dependent antifungal tolerance or full virulence.
  • The paper notes that the identity of the Crz1p post-translational modifications was not determined and that the precise NLS/NES and possible calcineurin docking motif require further analysis.
  • The paper acknowledges that virulence results differ from another study (Onyewu et al., 2004) and suggests possible explanations including strain background, URA3 positional effects, and starting inoculum; evident from the text, the microarray analysis used a single calcium concentration and three time points, so other conditions or targets may be missed.

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

Integrity signals

Registry

Crossref · 10.1111/j.1365-2958.2005.05037.x

Cites

58 of 58 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 29 cited sources checked · 0 supported · 0 flagged

  • Findingnone given

    The cnaΔ/Δ mutant was strongly avirulent in the same mice model.

    “which is in sharp contrast to the strong avirulence of cna D / D mutant in the same animal model.”
    Quote in the paperSubstantial claim
  • Findingown data

    Among 264 genes altered by at least twofold, upregulation of 60 genes was dependent on both calcineurin and CRZ1.

    “Among the expression of 264 genes altered by at least twofold, the upregulation of 60 genes was dependent on both calcineurin and CRZ1 .”
    Quote in the paperSubstantial claim
  • Findingown data

    Only CRZ1 was able to mediate calcium-dependent gene expression in S. cerevisiae.

    “and to mediate calcium-dependent gene expression in this yeast species.”
    Quote in the paperSubstantial claim
  • Findingown data

    Only CRZ1 was able to restore the calcium hypersusceptibility of a S. cerevisiae crz1Δ mutant.

    “Only CRZ1 was able to restore the calcium hypersusceptibility of a S. cerevisiae crz1 D mutant”
    Quote in the paperSubstantial claim
  • Findingown data

    A motif 5'-G(C/T)GGT-3' with similarity to the target sequence of Crz1p from S. cerevisiae was identified as a putative regulatory sequence in the upstream regions of calcineurin/Crz1p-dependent genes.

    “a motif [5 ¢ -G(C/T)GGT-3 ¢ ] with similarity to the target sequence of Crz1p (GNGGCG/TCA) from S. cerevisiae was identified as a putative regulatory sequence in the upstream regions of these cal-cineurin/Crz1p-dependent genes.”
    Quote in the paperSubstantial claim
  • May combine two claims. 38
  • Classified as probably not a substantial claim. 21

  • May combine two claims.

    Findingreasoning

    The three ORFs (orf19.7359, orf19.2356, orf19.9892) contain a C2H2 zinc finger domain, indicating they are putative transcription factors.

    “This domain was identified in the three different ORFs (orf19.7359,orf19.2356and orf19.9892)and therefore indicates that these ORFs are putative transcription factors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CRZ1 was not involved in tolerance to antifungal agents fluconazole and terbinafine, unlike calcineurin.

    “CRZ1 was not involved in tolerance to antifungal agents (fluconazole, terbinafine) on the opposite to calcineurin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In YEPD medium, the crz1Δ/Δ mutant and wild type showed similar colony appearance, in contrast to the cnaΔ/Δ mutant which formed wrinkled colonies.

    “In YEPD medium, the crz1∆/∆ mutant and the wild type showed similar colony appearance in contrast to the cna∆/∆ mutant which formed wrinkled colonies (Fig. 2, first row).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Fluconazole susceptibility was slightly increased by deletion of one or two CRZ1 copies compared with wild type but not to the extent of the cnaΔ/Δ mutant.

    “Fluconazole susceptibility was slightly increased by the deletion of one or two CRZ1 copies as compared with the wild type but not to the extent of the cna∆/∆ mutant (Fig. 1A, bottom, lanes 1, 2 and 4)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Crz1pΔC and Crz1pΔ232-322 were both localized constitutively in the nucleus.

    “Crz1p ∆C and Crz1p ∆232-322 were both localized constitutively in the nucleus (Fig. 3A)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The crz1Δ/Δ strain was affected in colony morphology compared with wild type but not to the same extent as the cnaΔ/Δ mutant.

    “the crz1∆/∆ strain was affected in colony morphology as compared with the wild type but not to the same extent as the cna∆/∆ mutant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the 69 calcium-activated and calcineurin-dependent genes with known or putative functions, five were involved in cell wall organization, three in cellular organization, five in cellular transport and homeostasis, seven in cell metabolism, and six in protein fate.

    “Among genes with known or putative functions, five were involved in cell wall organization (UTR2, ECM331, GSL21, IPF19968 and PMT2), three in cellular organization (AKR1, SAC6.5F and IPF13784), five in cellular transport and homeostasis (CCH1, RTA2, YIP3.3, ENA21.3 and VPS1), seven in cell metabolism and six in protein fate (see Table 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Kidneys from mice infected with wild type or CRZ1 revertant contained similar cfu (approximately 10^6 cfu g-1), whereas those from cnaΔ/Δ and crz1Δ/Δ mutants contained about 10^2 and 10^4 cfu g-1 respectively.

    “kidneys from mice infected with the wild type or the CRZ1 revertant contained similar colony-forming units (cfu) (approximately 10 6 cfu g -1 ), whereas those from the cna∆/∆ and crz1∆/∆ mutants contained about 10 2 and 10 4 cfu g -1 respectively (Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the presence of 10 μg ml-1 terbinafine, the crz1Δ/Δ mutant showed the same growth pattern as the wild type, whereas the cnaΔ/Δ mutant was not able to grow.

    “In the presence of 10 µg ml -1 terbinafine, the crz1∆/∆ mutant showed the same growth pattern as the wild type, whereas cna∆/∆ mutant was not able to grow (Fig. 1A, bottom, lanes 1, 2 and 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The region -384 to -363 of the UTR2 promoter contains a motif rich in guanine (5′-GGGCT-3′) conserved in the CDRE of S. cerevisiae and similar to the motif found in the promoters of the 60 calcineurin/Crz1p-dependent genes [5′-G(C/T)GGT-3′].

    “This region contains a motif rich in guanine (5′-GGGCT-3′) conserved in the CDRE of S. cerevisiae (Fig. 8, bottom) and similar to the motif found in the promoters of the 60 calcineurin/Crz1p-dependent genes [5′-G(C/T)GGT-3′] by genome-wide analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Time-kill curves with terbinafine showed the cnaΔ/Δ mutant was rapidly killed, while the crz1Δ/Δ mutant was still able to grow.

    “while the cna∆/∆ mutant was rapidly killed in the presence of terbinafine, the crz1∆/∆ mutant was still able to grow in the presence of this substance.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only CRZ1 but not CRZ2 from C. albicans restored wild-type phenotype in S. cerevisiae in the presence of calcium.

    “Only CRZ1 but not CRZ2 from C. albicans was restoring in S. cerevisiae a wild-type phenotype in the presence of calcium (Fig. S1 in Supplementary material)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The growth of the crz1Δ/Δ mutant was severely impaired by Ca2+ and Mn2+ but not Li+.

    “the growth of the crz1∆/∆ mutant was severely impaired by Ca 2+ , Mn 2+ but not Li + (Fig. 1A, top, lane 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only nine genes were strictly calcineurin-dependent and five genes were strictly Crz1p-dependent.

    “Only nine and five genes were strictly calcineurin-or Crz1pdependent respectively (Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    lacZ reporter activity was increased approximately by 3.5-fold in a wild-type strain after 2 h exposure to 200 mM of calcium, whereas this induction was decreased to 2.2-fold in a strain lacking CRZ1 and almost absent (1.1-fold) in the cna∆/∆ mutant.

    “As shown in Fig. 8 (top), lacZ reporter activity was increased approximately by 3.5-fold in a wild-type strain after 2 h exposure to 200 mM of calcium, whereas this induction was decreased to 2.2-fold in a strain lacking CRZ1 and almost absent (1.1-fold) in the cna∆/∆ mutant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The highest frequency of each base in the consensus sequence is: 95% G at position 1, 35% C or T at position 2, 75% G at position 3, 93% G at position 4, and 62% T at position 5.

    “The highest frequency of each base occurring at each position was shown: 95% of possible bases at the position 1 correspond to a G, 35% at the position 2 can be at equal probability a C or a T, 75% at the position 3 correspond to a G, 93% at the position 4 to a G and 62% at the position 5 to a T.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The number of genes with at least a twofold expression ratio reached a maximum at 20 min (176 genes), decreased at 40 min (82 genes) and increased at 60 min (162 genes).

    “the number of genes with at least a twofold expression ratio reached a maximum at 20 min (176 genes), decreased at 40 min (82 genes) and increased at 60 min (162 genes) (Fig. 6A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In cna∆/∆ and crz1∆/∆ mutant strains, the tested genes were not regulated by calcium, confirming their calcineurin/Crz1p-dependent regulation.

    “In cna∆/∆ and crz1∆/∆ mutant strains, the tested genes were not regulated by calcium and therefore confirmed their calcineurin/Crz1p-dependent regulation”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    The expression profiles of 264 calcium-regulated genes exhibiting a twofold or greater change were clustered with experiments carried out with the crz1∆/∆ or cna∆/∆ C. albicans strains at 20, 40 and 60 min time points.

    “The expression profiles of 264 calcium-regulated genes exhibiting a twofold or greater change were clustered with experiments carried out with the crz1∆/∆ or cna∆/∆ C. albicans strains at 20, 40 and 60 min time points (see Table2for experiment details).”
    Quote in the paperSubstantial claim

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

    Findingown data

    A set of genes is commonly regulated by calcineurin in the presence of calcium between S. cerevisiae and C. albicans.

    “List of common genes regulated by calcineurin in the presence of calcium between S. cerevisiae and C. albicans.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Virulence phenotypes between the crz1∆/∆ and cna∆/∆ mutants were not identical: while calcineurin is essential for virulence in a mice model of infection, CRZ1 has only a moderate effect.

    “Finally, virulence phenotypes between the crz1∆/∆ and cna∆/∆ mutants were not identical: while calcineurin is essential for virulence in a mice model of infection, CRZ1 has only a moderate effect (Fig. 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The crz1∆/∆ mutant could survive to terbinafine exposure as demonstrated by time-kill curves, in contrast with the lack of terbinafine tolerance of the cna∆/∆ mutant.

    “Moreover, the crz1∆/∆ mutant could survive to terbinafine exposure as demonstrated by time-kill curves (Fig. 2B), which is in strong contrast with the lack of terbinafine tolerance of the cna∆/ ∆ mutant as described (Sanglard et al., 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Sanglard et al., 2003

      Not checked in the free run.

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

    Findingnone given

    The CCH1 gene is present in the C. albicans genome and shares 55% of similarity with S. cerevisiae.

    “The CCH1 gene is present in the C. albicans genome and shares 55% of similarity with S. cerevisiae.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The crz1∆/∆ was able to grow on serum-containing agar, contrasting with the lack of survival of the cna∆/∆ in this medium.

    “In addition, the crz1∆/∆ was able to grow on serum-containing agar (Fig. 3), a feature contrasting with the lack of survival of the cna∆/∆ in this medium which was established earlier (Blankenship et al., 2003;Sanglard et al., 2003).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Blankenship et al., 2003

      Not checked in the free run.

    • cites Sanglard et al., 2003

      Not checked in the free run.

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

    Premisecitation

    The vast majority of calcineurin-dependent genes have Crz1p-dependent expression, suggesting Crz1p is a major mediator of calcineurin-activated gene expression in yeast.

    “The vast majority of calcineurin-dependent genes have a Crz1p-dependent expression, thus suggesting that Crz1p is a major mediator of calcineurin-activated gene expression in yeast (Yoshimoto et al ., 2002).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Yoshimoto et al., 2002

      Not checked in the free run.

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

    Premisecitation

    Crz1p contains a C2H2 zinc finger motif that binds to CDRE, which is sufficient for Ca2+- and calcineurin-dependent gene expression.

    “Crz1p contains a C 2 H 2 zinc finger motif that binds to a specific element on the promoter of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for Ca 2 + -and calcineurin-dependent gene expression (Stathopoulos and Cyert, 1997).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Stathopoulos and Cyert, 1997

      Not checked in the free run.

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

    Premisecitation

    Cyclosporin A and tracolimus (FK506) form complexes with cyclophilin A and FKBP12, respectively, which bind calcineurin and inhibit its enzymatic activity.

    “cyclosporin A and tracolimus form complexes with cyclophilin A and FKBP12 (FK506-binding protein 12) respectively.These immunosuppressant drugreceptor complexes bind calcineurin and inhibit its enzymatic activity (Liu et al ., 1991).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Liu et al., 1991

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    In S. cerevisiae, calcineurin dephosphorylates Crz1p, leading to its nuclear localization in response to increased Ca2+.

    “In response to increased Ca 2 + , activated calcineurin dephosphorylates Crz1p, thus leading to its nuclear localization (Stathopoulos-Gerontides et al ., 1999).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Stathopoulos-Gerontides et al., 1999

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In Candida albicans, the calcineurin pathway is involved in tolerance to antifungal agents, cation homeostasis and virulence.

    “In C. albicans , recent reports revealed the involvement of calcineurin in antifungal tolerance, cell morphogenesis and virulence.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Bader et al., 2003

      Not checked in the free run.

    • cites Blankenship et al., 2003

      Not checked in the free run.

    • cites Sanglard et al., 2003

      Not checked in the free run.

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

    Premisecitation

    Calcineurin is a Ca2+-calmodulin-dependent serine/threonine phosphatase consisting of a catalytic A subunit and a regulatory B subunit.

    “Calcineurin is a Ca 2 +calmodulin-dependent serine/threonine phosphatase consisting of a catalytic A subunit and a regulatory B subunit.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rusnak and Mertz, 2000

      Not checked in the free run.

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

    Premisecitation

    In S. cerevisiae, calcineurin activation induces 153 genes involved in cell wall biosynthesis, ion homeostasis, vesicle trafficking, lipid synthesis and protein degradation.

    “Using DNA microarrays, calcineurin activation induces 153 genes involved in cell wall biosynthesis, ion homeostasis, vesicle trafficking, lipid synthesis and protein degradation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yoshimoto et al., 2002

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Both CRZ1 and CNA regulate cation homeostasis.

    “Taken together, these observations indicated that both CRZ1 and CNA regulate cation homeostasis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Unlike the avirulence of the cnaΔ/Δ strain, CRZ1 was partially involved in virulence.

    “These findings demonstrate that, unlike the avirulence of the cna∆/∆ strain, CRZ1 was partially involved in virulence.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    CRZ1 has impact on colony morphology but only partial overlap with phenotypes dependent on calcineurin.

    “Taken together, these observations suggest that CRZ1 has impact on colony morphology, but also indicate only a partial overlap with phenotypes dependent on calcineurin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CRZ1 from C. albicans is the functional homologue of S. cerevisiae CRZ1 but not CRZ2.

    “the functional assays performed in S. cerevisiae strongly supported that CRZ1 from C. albicans is the functional homologue of CRZ1 but not CRZ2.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The obtained strains were verified by Southern blot for expected genomic integration and CRZ1 copy number.

    “The obtained strains were also verified by Southern blot for the expected genomic integration and CRZ1 copy number (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Crz1p is localized in the nucleus in the presence of calcium and this localization is dependent on calcineurin activity.

    “These results therefore suggest that Crz1p is localized in the nucleus in the presence of calcium and that this localization is dependent on calcineurin activity.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The phenotypes of strains constructed for virulence assays were identical to those of strains used in the first part of the study.

    “The phenotypes of strains constructed for virulence assays (cation and drug susceptibility, colony morphology) were identical to those of strains used in the first part of this study.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The C. albicans Cch1p could be a potential calcineurin target that could explain the fungicidal effect of fluconazole in a strain lacking calcineurin activity and therefore a fully functional CCS.

    “As Crz1p is not involved in the survival of C. albicans in the presence of antifungals, the C. albicans Cch1p could be a potential calcineurin target that could explain the fungicidal effect of fluconazole in a strain lacking calcineurin activity and therefore a fully functional CCS.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In S. cerevisiae, calcineurin has several substrates in addition to Crz1p not belonging to transcriptional activators.

    “In S. cerevisiae it is well known that calcineurin has several substrates in addition to Crz1p not belonging to transcriptional activators (Cyert, 2001;2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cyert, 2001;2003

      Not checked in the free run.

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

    Study design and conductnone given

    The animal experiments were carried out at the CHUV animal facility and were approved by the local ethical committee.

    “Animal experiments were carried out at the CHUV animal facility and were approved by the local ethical committee.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The significant threshold in expression ratio was determined by performing LOWESS normalization only on the TEF3 spots, TEF3 being a housekeeping gene, and considering the threshold above the most modulated TEF3 hybridization signals.

    “In order to determine a significant threshold in expression ratio, a LOWESS normalization was performed only on the TEF3 spots, TEF3 being an housekeeping gene.The significant threshold was considered above the most modulated TEF3 hybridization signals.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Dye swap controls were replaced by WTvsCNA and WTvsCRZ experiments in which cDNA from the wild type exposed to calcium was labelled with Cy-3 instead of Cy-5 as in the WT experiment.

    “Dye swap controls were replaced by WTvsCNA and WTvsCRZ experiments as described above in which cDNA from the wild type exposed to calcium was labelled with Cy-3 instead of Cy-5 as it is the case in the WT experiment (Table 2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The comparison suggests a species-specific calcineurin-dependent gene activation, which could result from the role of calcineurin in the adaptation of the organism to its environmental niche.

    “This comparison suggests a speciesspecific calcineurin-dependent gene activation, which could result from the role of calcineurin in the adaptation of the organism to its environmental niche.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In S. cerevisiae, Crz1p is shuffling from the cytoplasm to the nucleus after post-translational modifications dependent on calcineurin.

    “In this yeast species, Crz1p is shuffling from the cytoplasm to the nucleus after post-translational modifications dependent on calcineurin.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Santos and de Larrinoa, 2005

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    As in S. cerevisiae, calcineurin can act on other proteins by post-translational modifications in C. albicans.

    “As in S. cerevisiae, calcineurin can act on other proteins by post-translational modifications in C. albicans.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Calcineurin activity in C. albicans is required for multiple functions important for survival in the host, among them virulence and tolerance to antifungal agents.

    “As calcineurin activity in C. albicans is required for multiple functions important for survival in the host and among them the most relevant are virulence and tolerance to antifungal agents (Sanglard et al., 2003)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Sanglard et al., 2003

      Not checked in the free run.

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

    Study design and conductnone given

    Samples of the inoculum suspensions were plated onto YEPD agar to confirm the accuracy of the infection dose.

    “Samples of inoculum suspensions were plated onto YEPD agar to confirm the accuracy of the infection dose.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The C. albicans cDNA microarray covers nearly 98% of the total number of C. albicans genes and includes 27 control genes in each block, including negative controls (intergenic regions), crosshybridization controls (S. cerevisiae genes) and dynamic range controls by a serial dilution of the TEF3 gene from 1 to 1/32.

    “The microarray covers nearly 98% of the total number of C. albicans genes and includes 27 control genes in each block, including, for example, negative controls (intergenic regions), crosshybridization controls (S.cerevisiae genes) and dynamic range controls by a serial dilution of TEF3 gene from 1 to 1/32.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Recent microarray experiments performed in C. albicans under different pH conditions governed by the Rim101p-dependent pathway revealed that CRZ2 was downregulated by Rim101p in alkaline pH.

    “Recent microarray experiments performed in C. albicans under different pH conditions governed by the Rim101pdependent pathway revealed that CRZ2 was downregulated by Rim101p in alkaline pH (Bensen et al., 2004).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bensen et al., 2004

      Not checked in the free run.

50 of 50

  • The paper relies on this without support.

    The components of the calcineurin pathway are still poorly investigated in C. albicans.

    “However, the components of the calcineurin pathway are still poorly investigated in this yeast species.”

  • The authors state this as an assumption.

    The NLS is localized between residues 2 and 207 of Crz1p.

    “it can be assumed that the NLS is localized between residues 2 and 207.”

  • The paper relies on this without support.

    Calcium activates the calcineurin signalling pathway in C. albicans.

    “As calcium is a classical activator of calcineurin activity, this cation was used as an effector to mimic the activation of the calcineurin signalling pathway in C. albicans.”

  • The paper relies on this without support.

    The C. albicans cdr1∆/∆ cdr2∆/∆ mutant strain (DSY1025) allows optimal oestradiol-inducible transcriptional activation of the different fusion proteins.

    “All plasmids were used to transform a C. albicans cdr1∆/∆ and cdr2∆/∆ mutant strain (DSY1025) to allow optimal oestradiol-inducible transcriptional activation of the different fusion proteins.”
4 of 4

Claims other papers make about this one

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

  • Crz1 is the key mediator of calcineurin-dependent transcriptional responses and is implicated in tolerance to azoles in both S. cerevisiae and C. albicans.

    “Crz1 is the key mediator of calcineurin-dependent transcriptional responses [37],[41] and is implicated in tolerance to azoles in both S. cerevisiae and C. albicans [20],[42].”

    From Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin (2009) · cites this paper as [37]

    The Abstract says that, unlike calcineurin, CRZ1 was not involved in tolerance to fluconazole or terbinafine in C. albicans. The phenotypic-analysis section reports only a slight change in fluconazole susceptibility after CRZ1 deletion, far less than after calcineurin deletion. This contradicts the cited azole-tolerance claim for C. albicans, although the paper does support a role for Crz1 in calcineurin-dependent gene expression. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • The UTR2-lacZ reporter is activated by calcium chloride and reports calcineurin activation.

    “As expected, exposure of cells containing the UTR2-lacZ reporter to calcium chloride resulted in activation of calcineurin relative to the untreated control (P<0.001, ANOVA, Bonferroni's Multiple Comparison Test Figure 4C).”

    From Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin (2009) · cites this paper as [37]

  • Calcineurin also promoted tolerance to terbinafine in C. albicans, to tunicamycin and dithiothreitol in S. cerevisiae, and to SDZ 90-215 in all these species.

    “Calcineurin also promoted tolerance to terbinafine in C. albicans (58), to tunicamycin and dithiothreitol in S. cerevisiae (59, 60), and to SDZ 90-215 in all these species (61).”

    From Echinocandin tolerance and persistence <i>in vitro</i> are regulated by calcineurin signaling in <i>Candida glabrata</i> (2026) · cites this paper as [58]

  • The Δcrz1 mutation has little effect on virulence in Candida albicans.

    “this mutation has little effect on virulence in both C. albicans (14, 23) and C. glabrata (Fig. 3).”

    From Roles of Calcineurin and Crz1 in Antifungal Susceptibility and Virulence of <i>Candida glabrata</i> (2010) · cites this paper as [14]

  • Crz1 is a downstream effector of calcineurin and is involved in azole tolerance in Candida albicans.

    “The transcription factor Crz1 is a downstream effector of calcineurin and is involved in azole tolerance in C. albicans (14, 23, 28);”

    From Roles of Calcineurin and Crz1 in Antifungal Susceptibility and Virulence of <i>Candida glabrata</i> (2010) · cites this paper as [14]

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.

  • Reported absence of cna∆/∆ cells contradicts the spleen measurement

    Major

    The text says no cna∆/∆ cells were recovered from either organ on day 3, but Table 1 reports 3.3 ± 0.1 log cfu g -1 in spleens on that day.

  • UTR2 promoter coordinates do not span 24 bp

    Minor

    Positions -384 through -363 span 22 bases when both endpoints are included, rather than the stated 24 bp.

    Reported 24 · recomputed 21

  • UTR2 flanking fragment is longer than the stated 500 bp

    Minor

    The stated coordinates for the 3′ flanking fragment differ by 550 bp, not 500 bp (or span 551 positions if both endpoints are included).

    Reported 500 · recomputed 550

3 of 3

Cites

Flagged first, then by citations

2 of the 60 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Hanahan D., 1985, DNA Cloning. A Practical Approach, 109

Cited by

Sorted by citations

Related

Authors
Karababa, M., Valentino, E., Pardini, G., Coste, A. T., Bille, J., & Sanglard, D.
Affiliations
Centre Hospitalier Universitaire Vaudois (CH)
Corresponding author
Sanglard, Dominique
ORCID
Venue
Molecular Microbiology
Publisher
Wiley
Published
2006-01-20
Volume
59
Issue
5
Pages
1429-1451
ISSN
  • 0950-382X (ISSN-L)
  • 1365-2958
DOI
10.1111/j.1365-2958.2005.05037.x
OpenAlex
W1557800963
Semantic Scholar
a3d8b4bef12bfb3e470306f4cf4502a61002bff7
Cited by
231
References
60
Citations by year
2026: 13 · 2025: 6 · 2024: 3 · 2023: 11 · 2022: 15 · 2021: 14 · 2020: 11 · 2019: 6 · 2018: 10 · 2017: 11 · 2016: 11 · 2015: 7 · 2014: 16 · 2013: 9 · 2012: 15
FWCI
6.35
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations
  • Molecular Microbiology (publishedVersion)
  • PubMed (publishedVersion)
  • IRIS (submittedVersion)
  • SERVAL (Université de Lausanne) (submittedVersion)
Funders
Howard Hughes Medical Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
Keywords
Crz1, calcineurin, Candida albicans, calcium signaling, transcriptional regulation, C2H2 zinc finger, antifungal drug tolerance, cation homeostasis, virulence, nuclear localization, gene expression, Saccharomyces cerevisiae model, fluconazole resistance
MeSH terms
Animals, Antifungal Agents, Calcium, Candida albicans, Candidiasis, Cell Nucleus, Female, Fungal Proteins, Glycoside Hydrolases, Mice, Inbred BALB C, Mutation, Regulatory Sequences, Nucleic Acid, Virulence, Signal Transduction, Gene Expression Regulation, Fungal, Zinc Fingers, Genome, Fungal, Calcineurin, Saccharomyces cerevisiae Proteins, Intracellular Signaling Peptides and Proteins, 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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