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Cited by 78Fungal and yeast genetics researchAntifungal resistance and susceptibilitySignaling Pathways in Disease

Functional characterization of the Candida albicans CRZ1 gene encoding a calcineurin-regulated transcription factor

Open access
Santos, Mikel, de Larrinoa, Iñigo F.·2005·Current Genetics
DOI 10.1007/s00294-005-0003-8
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Abstract

Calcineurin is a phosphoprotein phosphatase devoted to the transduction of Ca(2+)-signals in eukaryotes. In the human pathogen Candida albicans, calcineurin function is required for cell morphogenesis, azole tolerance, membrane stress responses, survival in serum and virulence in mice. Molecular mechanisms as well as targets downstream C. albicans calcineurin involved in all these processes are still uncharacterized. Here we report the characterization of a C. albicans Crz1 calcineurin-regulated transcription factor using a Saccharomyces cerevisiae crz1Delta defective strain as heterologous host. CaCrz1p fulfils the function of its S. cerevisiae homolog protein to control the expression of several Ca(2+)/calcineurin-responsive genes acting on the CDRE sequence in promoters. In the model yeast, CaCrz1p activity and localization are regulated by calcineurin. Deletion of CRZ1 gene renders C. albicans hypersensitive to alkaline cations and membrane stress conditions, including that elicited by SDS and antifungal azoles. Our findings indicate that CaCrz1p is member of a calcium-regulated pathway required for the maintenance of membrane integrity.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper characterizes *Candida albicans* ORF19.7359 as *CaCRZ1*, encoding a calcineurin-regulated Crz1 homolog that can functionally replace *S. cerevisiae* Crz1p in a heterologous *crz1Δ* host. Deletion of *CaCRZ1* renders *C. albicans* hypersensitive to alkaline cations and membrane stress, including SDS and antifungal azoles.

Key findings

  • *C. albicans* ORF19.7359 complements *S. cerevisiae crz1Δ* growth defects in high Na⁺, Mn²⁺, and Ca²⁺, and its overexpression suppresses Na⁺/Mn²⁺ sensitivity in a calcineurin-defective *cnb1Δ* strain, similar to *ScCRZ1*.
  • CaCrz1p regulates calcineurin-dependent LacZ reporters: PMR1-LacZ induction by Ca²⁺ is blocked by FK506; PMC1-LacZ induction by CaCRZ1 is much weaker than by ScCRZ1 (e.g., at 3 h, 34 ± 2.7 vs. 680 ± 33 units), while control remains <1; CDRE ENA1-LacZ shows a twofold increase in noninduced conditions and is abolished when the CDRE is mutated.
  • CaCrz1-GFP is cytosolic in noninduced cells, translocates to the nucleus after Ca²⁺ addition, and this translocation is blocked by FK506 and absent in a *cnb1Δ* strain.
  • A *C. albicans crz1/crz1* null mutant is viable but hypersensitive to Ca²⁺, Li⁺, Mn²⁺, 0.025% SDS, fluconazole, and miconazole; reintegration of wild-type *CaCRZ1* restores the wild phenotype. The *crz1/crz1* mutant is intermediate in Li⁺ sensitivity between wild type and a *cmp1/cmp1* calcineurin mutant.

Methods

  • Heterologous *S. cerevisiae crz1Δ* and *cnb1Δ* strains were used to test *CaCRZ1* complementation, ion stress phenotypes, and LacZ reporter expression from PMR1, PMC1, and native/mutated CDRE ENA1 promoters.
  • CaCrz1p localization was assayed with a CaCRZ1-GFP reporter by fluorescence microscopy before and after 0.2 M CaCl₂, with FK506 pretreatment and in a *cnb1Δ* background.
  • *C. albicans crz1/crz1* mutants were constructed in CAI-4 by sequential URA3-dpl200 disruption, confirmed by Southern blotting, and phenotyped by spot dilution assays on YPD plates with cations, SDS, fluconazole, or miconazole.

Limitations

  • The paper states that its Li⁺ hypersensitivity result for *crz1/crz1* differs from Onyewu et al. (2004), and suggests the choice of experimental conditions enhancing cation sensitivity may explain the discrepancy.
  • The paper notes that the integrative PMC1-LacZ reporter was below the β-galactosidase assay threshold, so a multicopy reporter was used, and that CaCRZ1-dependent PMC1 induction showed slower kinetics and was far weaker than ScCRZ1-dependent induction.
  • Evident from the text: much of the functional characterization relies on a heterologous *S. cerevisiae* host rather than native *C. albicans*, and the study does not directly test virulence or serum survival despite referencing those calcineurin-dependent processes.

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

Integrity signals

Registry

Crossref · 10.1007/s00294-005-0003-8

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Both ORF19.2356 and ORF19.7359 exhibit C-terminal structural motifs highly homologous to those in the S. cerevisiae Crz1 and S. pombe Prz1 proteins.

    “Near their C-terminal end, both sequences exhibit structural motifs highly homologous (identities 52% and positives 65% for ORF19.2356; and 52% and 69% respectively for ORF19.7359) to those found in the S. cerevisiae Crz1 and S. pombe Prz1 proteins (Fig. 1a).”
    Quote in the paperSubstantial claim
  • Findingown data

    Induction of the PMR1-LacZ reporter is no longer observed if cells are treated with FK506 shortly before calcium addition.

    “Moreover, induction of the reporter is no longer observed if cells are treated with the calcineurin inhibitory drug FK506 shortly before the addition of calcium to the culture (Fig. 3b).”
    Quote in the paperSubstantial claim
  • Findingown data

    Both ScCRZ1 and the C. albicans homolog genes cause a similar improvement in Na+ and Mn2+ tolerance when transformed into cnb1D cells.

    “The photographs in Fig. 2c show that both ScCRZ1 and the C. albicans homolog genes cause a similar effect of improved Na + and Mn 2+ tolerance after transformation in cnb1D cells.”
    Quote in the paperSubstantial claim
  • Findingown data

    Integrative PMC1-LacZ reporter expression was below the threshold in the beta-galactosidase activity assay.

    “Integrative PMC1-LacZ reporter expression was below the threshold in b-galactosidase activity enzymatic assay and our PMC1 expression monitoring effort was fruitless.”
    Quote in the paperSubstantial claim
  • Findingown data

    The putative CaCRZ1 gene improved growth of the S. cerevisiae crz1D mutant in high Na+ and Mn2+ conditions.

    “Phenotypic analysis in growth-limiting Ca 2+ , Mn 2+ and Na + concentrations showed that the putative CaCRZ1 gene clearly improved yeast growth in high Na + and Mn 2+ conditions for the crz1D mutant (Fig. 2a).”
    Quote in the paperSubstantial claim
  • May combine two claims. 18
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    In high calcium conditions, the positive effect of the putative CaCRZ1 gene on crz1D growth is faint after 48 h but becomes evident three days later.

    “In high calcium conditions the positive effect on cell growth is faint after 48 h, but it becomes evident three days later (Fig. 2b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The alignment of ORF19.7359 with SpPrz1p extends along the complete SpPrz1 sequence, whereas only partial sequence alignments were found for ORF19.2356.

    “While only partial sequence alignments were found for ORF19.2356, the alignment of ORF19.7359 and SpPrz1p extended along the complete SpPrz1 sequence ranging from N-to C-terminus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The C. albicans crz1/crz1 null mutant is viable and displays no growth defects in YPD medium at 30°C.

    “The C. albicans crz1/crz1 null mutant is viable and displays no growth defects in YPD medium at 30°C”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After calcium addition to the culture, CaCrz1-GFP translocates to the nucleus and rapidly accumulates there.

    “After calcium addition to cells the GFP reporter translocates to the nucleus and a fast nuclear CaCrz1-GFP protein accumulation can be observed.”
    Quote in the paperSubstantial claim

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

    Findingnone given

    The C. albicans genome contains two putative Crz1p homolog proteins bearing zinc finger motifs which exhibit high homology to the S. cerevisiae Crz1 protein carboxyl-terminal DNA binding domain.

    “The C. albicans genome contains two putative Crz1phomolog proteins bearing zinc finger motifs which exhibit high homology to the S. cerevisiae Crz1 protein carboxyl-terminal DNA binding domain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The CaCrz1p effect on CDRE ENA1-LacZ expression is observed in noninduced conditions as a twofold increase and in Ca2+-induced conditions.

    “This CaCrz1p effect on CDRE ENA1 -LacZ expression is observed either in noninduced (twofold increase) and Ca 2+ -induced condi-”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Defective prz1 and crz1 yeasts exhibit hypersensitivity to calcium ions, consistent with decreased expression of the Pmc1 Ca2+-pump.

    “Like CRZ1, Prz1+ gene is not required for cell growth in standard conditions but defective prz1and crz1-yeasts exhibit hypersensitivity to calcium ions, consistent with a decreased expression of the Pmc1 Ca 2+ -pump.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hirayama et al. 2003 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    In fungi, calcineurin mediates responses to environmental stress conditions that compromise cell viability.

    “The characterization of this enzyme in genetically tractable yeasts has revealed a major role for calcineurin in fungi as mediator of responses to environmental stress conditions that compromise cell viability (reviewed in Kraus and Heitman 2003;Cyert 2003;Sugiura et al. 2002).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Kraus and Heitman 2003 source

      Not checked in the free run.

    • cites Cyert 2003 source

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    • cites Sugiura et al. 2002 source

      Not checked in the free run.

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

    Premisecitation

    S. cerevisiae cells lacking calcineurin are hypersensitive to Na+, Li+, Mn2+ and hydroxyl ions.

    “However, S. cerevisiae cells lacking this phosphatase are hypersensitive to some ions including Na + , Li + , Mn 2+ and hydroxyl ions (Nakamura et al. 1993;Breuder et al. 1994;Mendoza et al. 1994;Farcasanu et al. 1995)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Nakamura et al. 1993 source

      Not checked in the free run.

    • cites Breuder et al. 1994 source

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    • cites Mendoza et al. 1994 source

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  • May combine two claims.

    Interpretationown data

    The cloned C. albicans sequences suppress ionic sensitivities of the crz1D strain, indicating that the C. albicans ORF19.7359-encoding gene restores CRZ1 function in crz1D yeast.

    “The finding that the cloned C. albicans sequences suppress ionic sensitivities displayed by the crz1D strain indicates that the C. albicans ORF19.7359encoding gene restores CRZ1 gene function in a crz1D defective yeast.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    C. albicans mutants lacking either calcineurin subunit are viable but exhibit increased sensitivity to ionic stress conditions.

    “C. albicans mutants lacking either one of the calcineurin subunits are viable but exhibit increased sensitivity to ionic stress conditions (Cruz et al. 2002;Sanglard et al. 2003;Bader et al. 2003).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Cruz et al. 2002 source

      Not checked in the free run.

    • cites Sanglard et al. 2003 source

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    • cites Bader et al. 2003 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A homozygous C. albicans mutant lacking a CRZ1-homolog gene has been described, and many differences in phenotypes between this mutant and the calcineurin-defective cnb1/cnb1 strain have been reported.

    “a homozygous C. albicans mutant lacking a CRZ1-homolog gene has been recently described and many differences in the phenotypes of this mutant and the calcineurin-defective cnb1/cnb1 strain have been reported (Onyewu et al. 2004).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Onyewu et al. 2004 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Calcineurin is essential for C. albicans survival in serum and virulence in a murine model of infection.

    “Calcineurin has been shown to be essential for C. albicans survival in serum and virulence in a murine model of infection, but the molecular mechanisms involved are still unknown (Blankenship et al. 2003;Bader et al. 2003;Sanglard et al. 2003).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Blankenship et al. 2003 source

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    • cites Bader et al. 2003 source

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    • cites Sanglard et al. 2003 source

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  • May combine two claims.

    Premisecitation

    Crz1p activity and subcellular localization are controlled by calcineurin.

    “Crz1p activity and subcellular localization are accurately controlled by calcineurin (Stathopoulos-Gerontides et al. 1999).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Stathopoulos-Gerontides et al. 1999 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Calcineurin-dependent expression of ion transporting ATPases and a glucan synthase is regulated by the ScCrz1/Tcn1/Hal8 transcription factor.

    “Calcineurin-dependent expression of these genes is regulated by the ScCrz1/Tcn1/Hal8 transcription factor (Stathopoulos and Cyert 1997;Matheos et al. 1997;Mendizabal et al. 1998)”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Stathopoulos and Cyert 1997 source

      Not checked in the free run.

    • cites Matheos et al. 1997 source

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    • cites Mendizabal et al. 1998 source

      Not checked in the free run.

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

    Premisecitation

    Calcineurin function is essential for some yeast mutants with cell wall defects.

    “and calcineurin function is essential for some yeast mutants with cell wall defects (Garret-Engele et al. 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Garret-Engele et al. 1995

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Crz1p binds to the CDRE sequence in FKS2 and ENA1 promoters and activates expression of calcineurin-responsive genes.

    “To drive this transcriptional stress response, Crz1p binds to a cognate sequence called CDRE, already identified in FKS2 and ENA1 gene promoters, and activates the expression of calcineurin-responsive genes (Stathopoulos and Cyert 1997;Mendizabal et al. 2001).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Stathopoulos and Cyert 1997 source

      Not checked in the free run.

    • cites Mendizabal et al. 2001 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    CaCrz1p functions as a transcription factor to control Ca2+-induced PMR1 expression in a calcineurin-dependent fashion, and calcineurin inhibition results in a CaCrz1p transcriptional activity knock-out.

    “that CaCrz1p functions as a transcription factor to control Ca 2+ induced PMR1 expression in a calcineurin-dependent fashion and calcineurin inhibition results in the CaCrz1p transcriptional activity knock-out.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    ScCrz1 is the main effector mediating all known calcineurin-dependent transcriptional changes in S. cerevisiae.

    “which, in genome-wide expression studies using DNA microarrays, has been identified as the main effector mediating all known calcineurin-dependent transcriptional changes in S. cerevisiae (Yoshimoto et al. 2002).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yoshimoto et al. 2002 source

      Not checked in the free run.

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

    Premisecitation

    Calcineurin is a serine/threonine-specific protein phosphatase specialized in the transduction of Ca2+-signals in eukaryotes.

    “Calcineurin is a serine and threonine-specific protein phosphatase specialized in the transduction of Ca 2+ -signals in eukaryotes (Aramburu et al. 2004).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Aramburu et al. 2004 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The related yeast S. cerevisiae and C. albicans diverged more than 140 million years ago, but primary biological processes as well as major effectors governing them are still conserved in both organisms.

    “The related yeast S. cerevisiae and C. albicans diverged more than 140 million years ago, but primary biological processes as well as major effectors governing them are still conserved in both organisms (Berman and Sudbery 2002).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Berman and Sudbery 2002 source

      Not checked in the free run.

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

    Premisecitation

    Several studies in fungi have elucidated a role for calcium signaling in response to membrane stress, including that elicited by detergents and antifungal azoles.

    “Several studies in fungi have elucidated a role for calcium signaling in response to membrane stress, including that elicited by detergents and antifungal azoles (Edlind et al. 2002;Kaur et al. 2004).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Edlind et al. 2002 source

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    • cites Kaur et al. 2004 source

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  • Classified as probably not a substantial claim.

    Study design and conductown data

    The identity and fidelity of the cloned C. albicans CRZ1 fragment was verified by sequencing.

    “The identity and fidelity of the cloned fragment was verified by sequencing.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Data represent the average of three independent transformants, with standard deviations less than 15% for multicopy and less than 10% for integrative plasmids.

    “Data represent the average of three independent transformants.The standard deviations of average values were less than 15% for multicopy and less than 10% for integrative plasmids.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    CRZ1 gene deletions were checked by PCR and crz1D strains were confirmed in phenotypic assays.

    “CRZ1 gene deletions were checked by PCR with the above-mentioned primers, and crz1D strains were confirmed in phenotypic assays.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    CaPMC1 is a Ca2+-calcineurin-responsive gene having a CDRE-like sequence in its promoter, and calcineurin-dependent CaPMC1 induction in high calcium has been reported in C. albicans cna1/cna1 mutant.

    “Previous work in the C. albicans cna1/cna1 mutant has shown that CaPMC1 is a Ca 2+ -calcineurin-responsive gene having a CDRE-like sequence in its promoter, and a calcineurindependent CaPMC1 induction in high calcium has been reported (Sanglard et al. 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Sanglard et al. 2003 source

      Not checked in the free run.

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

    Study design and conductown data

    Two independent clones from each mutant strain were checked by Southern hybridization and used for phenotypic assays.

    “Two independent clones from each mutant strain were checked by Southern hybridization and used for phenotypic assays (Fig. 6b).”
    Quote in the paperSubstantial claim
27 of 27

  • The paper relies on this without support.

    Molecular mechanisms and targets downstream of C. albicans calcineurin involved in cell morphogenesis, azole tolerance, membrane stress responses, survival in serum and virulence in mice are uncharacterized.

    “Molecular mechanisms as well as targets downstream C. albicans calcineurin involved in all these processes are still uncharacterized.”

  • The paper relies on this without support.

    The subcellular localization of the CaCrz1-GFP fusion protein reflects that of native CaCrz1p.

    “the CaCrz1-GFP protein localization was investigated by fluorescence microscopy before and after calcium addition to the culture.”
2 of 2

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References 49 · Cited by 78

Authors
Santos, M., & de Larrinoa, I. F.
Venue
Current Genetics
Publisher
Springer Science and Business Media LLC
Published
2005-07-26
Volume
48
Issue
2
Pages
88-100
ISSN
  • 0172-8083 (ISSN-L)
  • 1432-0983
DOI
10.1007/s00294-005-0003-8
OpenAlex
W1996580638
Semantic Scholar
032bfe7010006f12261e11b8bc906c30da58f706
Cited by
78
References
49
Citations by year
2026: 3 · 2025: 2 · 2023: 2 · 2022: 5 · 2021: 5 · 2020: 3 · 2019: 2 · 2018: 1 · 2017: 3 · 2016: 4 · 2015: 2 · 2014: 4 · 2013: 2 · 2012: 7
FWCI
2.74
Type
journal article
Language
en
Open access
Licence
Locations
  • Current Genetics (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Eusko Jaurlaritza (IE019)
  • Euskal Herriko Unibertsitatea
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
Keywords
Candida albicans, calcineurin, calcium signaling, membrane stress response, SDS sensitivity, membrane integrity, heterologous expression in yeast, fungal virulence
MeSH terms
Binding Sites, Candida albicans, Cell Membrane, Fungal Proteins, Promoter Regions, Genetic, Saccharomyces cerevisiae, Transcription Factors, Calcineurin

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