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High Resistance to Oxidative Stress in the Fungal Pathogen Candida glabrata Is Mediated by a Single Catalase, Cta1p, and Is Controlled by the Transcription Factors Yap1p, Skn7p, Msn2p, and Msn4p

Mayra Cuéllar-Cruz, Marcela Briones-Martin-del-Campo, Israel Cañas-Villamar, Javier Montalvo-Arredondo, Lina Riego-Ruiz, Irene Castaño, Alejandro De Las Peñas
Mayra Cuéllar-Cruz
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Marcela Briones-Martin-del-Campo
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Israel Cañas-Villamar
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Javier Montalvo-Arredondo
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Lina Riego-Ruiz
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Irene Castaño
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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Alejandro De Las Peñas
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, San Luis Potosí 78216, México
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  • For correspondence: cano@ipicyt.edu.mx
DOI: 10.1128/EC.00011-08
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  • FIG. 1.
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    FIG. 1.

    C. glabrata, C. albicans, and S. cerevisiae LP resistance to H2O2. Saturated cultures of C. glabrata (C.g.) strain BG14 and CI MC7 and MC22 (A), C. albicans (C.a.) strain CAI4 and CI CA5 and CA7 (B), and S. cerevisiae (S.c.) strain W303 and CI YJM128 and YJM336 (C) were diluted with fresh medium (YPD) so that all strains reached an OD600 of 0.5 after seven doublings at 30°C. C. glabrata and C. albicans strains were divided and exposed to 0, 10, 20, 30, 40, 50, and 100 mM H2O2 and S. cerevisiae strains were exposed to 2, 4, 6, and 8 mM H2O2 for 3 h. For adaptation experiments, C. glabrata and C. albicans cells were pretreated for 1 h with 10 mM H2O2 and then with 100 mM H2O2 for 2 additional hours. After the treatment, H2O2 was removed by centrifugation. The cultures were resuspended in distilled water, and the OD600s were adjusted when needed to 0.5. Cultures were serially diluted, and each dilution was spotted onto YPD plates, ensuring that the same amounts of cells were plated. Plates were incubated at 30°C. See Materials and Methods.

  • FIG. 2.
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    FIG. 2.

    C. glabrata, C. albicans, and S. cerevisiae SP resistance to H2O2. Saturated cultures of C. glabrata (C.g.) strain BG14 and CI MC7 and MC22 (A), C. albicans (C.a.) strain CAI4 and CI CA5 and CA7 (B), and S. cerevisiae (S.c.) strain W303 and CI YJM128 and YJM336 (C) were diluted to an OD600 of 0.5 with spent medium from the same cultures. The cells were divided into aliquots and treated for 3 h with H2O2 at different concentrations: for C. glabrata, 500, 800, 1,000, and 1,500 mM; for C. albicans, 0, 50, 100, 300, and 500 mM; and for S. cerevisiae, 0, 10, 50, 100, and 200 mM. After the treatment, the cultures remained at an OD600 of 0.5, oxidant was removed by centrifugation, cells were resuspended in distilled water, and suspensions were diluted and spotted onto YPD plates. Plates were incubated at 30°C. See Materials and Methods.

  • FIG. 3.
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    FIG. 3.

    Regulation of the OSR to H2O2 in LP. The wt (BG14) and single, double, and triple mutants with yap1Δ, skn7Δ, msn2Δ, and msn4Δ mutations were grown and treated with H2O2 as described in the legend to Fig. 1. See Materials and Methods.

  • FIG. 4.
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    FIG. 4.

    Regulation of the OSR to H2O2 in SP. The wt (BG14) and single, double, and triple mutants with yap1Δ, skn7Δ, msn2Δ, and msn4Δ mutations were grown and treated with H2O2 as described in the legend to Fig. 2. See Materials and Methods.

  • FIG. 5.
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    FIG. 5.

    ScCta1p and CgCta1p alignment. ScCta1p and CgCta1p are 85% similar across the entire lengths of the proteins. Identical residues are boxed and shaded. SKF (indicated by boxes and asterisks) is the peroxisomal targeting signal.

  • FIG. 6.
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    FIG. 6.

    Analysis of Cta1p in the OSR. Experiments with both LP and SP wt (BG14), cta1Δ, yap1Δ skn7Δ msn4Δ, and yap1Δ skn7Δ msn2Δ msn4Δ cells were performed as described in the legends to Fig. 1 and 2. See Materials and Methods.

  • FIG. 7.
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    FIG. 7.

    Growth curves of the cta1Δ mutant versus the wt (BG14) at 37°C.

  • FIG. 8.
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    FIG. 8.

    Cta1p is dispensable in vivo. Numbers of CFU in kidney, spleen, and liver tissues 7 days postinfection with the wt (BG462) and the cta1Δ mutant (CGM351) are shown. Individual datum points represent results for individual mice in groups of 10 mice. The bars indicate the geometric mean for each group. See Materials and Methods.

Tables

  • Figures
  • TABLE 1.

    Strains used in this study

    StrainParentGenotype and/or descriptionReference or source
    E. coli strains
        DH10BF−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara,leu)7697 galU galK λ−rpsL nupG 7
        JM109 recA1 endA1 gyrA96 thi hsdR17(rK− mK+) relA1 supE44 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] 77
    S. cerevisiae strains
        W303 MAT a ura3-1 leu2-3,112 his3-11,15 trp1-1 can1-100 ade2-1 ade3::hisG 51
        YJM128Clinical isolate 12
        YJM336Clinical isolate 12
    C. albicans strains
        CAI4 ura3Δ::imm434/ura3Δ::imm434 25
        CA5Clinical isolateLab collection
        CA7Clinical isolateLab collection
    C. glabrata strains
        MC7Clinical isolateLab collection
        MC22Clinical isolateLab collection
        BG2Clinical isolate (strain B) 22
        BG14BG2 ura3Δ::Tn903 G418r; wt Ura− strain used in this study 15
        BG462BG14 URA3 18
        BG1739 (msn2Δ mutant)BG14 ura3Δ::Tn903 G418rmsn2Δ Hygs Ura− pRD96/BcgIR. Domergue and B. Cormack
        BG1740 (msn4Δ mutant)BG14 ura3Δ::Tn903 G418rmsn4Δ Hygs Ura− pRD97/BcgIR. Domergue and B. Cormack
        BG1742 (msn2Δ msn4Δ mutant)BG1739 ura3Δ::Tn903 G418rmsn2Δ msn4Δ::hph HygrR. Domergue and B. Cormack
        CGM295 (cta1Δ mutant)BG14 ura3Δ::Tn903 G418rcta1Δ::hph Hygr pCV15/BcgIThis work
        CGM297 (yap1Δ mutant)BG14 ura3Δ::Tn903 G418ryap1Δ::hph Hygr pCV17/BcgIThis work
        CGM306 (skn7Δ mutant)BG14 ura3Δ::Tn903 G418rskn7Δ::hph Hygr pCV21/BcgIThis work
        CGM308 (yap1Δ mutant)CGM297 ura3Δ::Tn903 G418ryap1Δ Hygs Ura−This work
        CGM310 (yap1Δ skn7Δ mutant)CGM308 ura3Δ::Tn903 G418ryap1Δ skn7Δ::hph HygrThis work
        CGM351 (cta1Δ mutant)CGM295 URA3 cta1Δ::hph HygrThis work
        CGM385 (yap1Δ skn7Δ mutant)CGM310 ura3Δ::Tn903 G418ryap1Δ skn7Δ Hygs Ura−This work
        CGM386 (yap1Δ skn7Δ msn2Δ mutant)CGM385 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn2Δ::hph HygrThis work
        CGM388 (yap1Δ skn7Δ msn4Δ mutant)CGM385 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ::hph HygrThis work
        CGM480 (yap1Δ skn7Δ msn4Δ mutant)CGM388 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ Hygs Ura−This work
        CGM537 (yap1Δ skn7Δ msn4Δ msn2Δ mutant)CGM480 ura3Δ::Tn903 G418ryap1Δ skn7Δ msn4Δ msn2Δ::hph HygrThis work
  • TABLE 2.

    Plasmids used in this study

    PlasmidDescription and/or relevant genotypeReference or source
    Plasmids for cloning, construction, and marker removal
        pGEM-TCloning vector; Kmr AprPromega
        pAP599Cloning vector with 2 FRT direct repeats flanking the hygromycin marker [FRT-PPGK::hph::(3′ UTR of HIS3)-FRT] for construction of multiple mutants; URA3 Hygr Ampr 19
        pMZ17Replicative vector expressing ScFLP1 (recombinase gene) for removing the hygromycin marker; PPGK::FLP1::(3′ UTR of HIS3) CgCEN ARS AmprCormack lab collection
        pBC34.1pUC19::CgURA3; 2.2-kb PstI fragment; Apr 15
    Plasmids for deletion
        pCV15 cta1ΔA 0.906-kb SpeI/BamHI PCR fragment (corresponding to primers 57 and 58) carrying the promoter region of CTA1 and a 0.682-kb HindIII/KpnI PCR fragment (corresponding to primers 60 and 61) carrying the 3′ UTR of CTA1 were cloned into pAP599 AprThis work
        pCV17 yap1ΔA 0.846-kb XhoI/HindIII PCR fragment (corresponding to primers 7 and 8) carrying the promoter region of YAP1 and a 0.652-kb BamHI/SacI PCR fragment (corresponding to primers 11 and 12) carrying the 3′ UTR of YAP1 were cloned into pAP599 AprThis work
        CV21 skn7ΔA 0.875-kb KpnI/XhoI PCR fragment (corresponding to primers 4 and 5) carrying the promoter region of SKN7 and a 0.929-kb BamHI/SacI PCR fragment (corresponding to primers 1 and 2) carrying the 3′ UTR of SKN7 were cloned into pAP599 AprThis work
        pRD96 msn2ΔA 0.691-kb KpnI/XhoI PCR fragment (corresponding to primers 2984 and 2985) carrying the promoter region of MSN2 and a 0.520-kb BamHI/SacI PCR fragment (corresponding to primers 2986 and 2987) carrying the 3′ UTR of MSN2 were cloned into pAP599 AprR. Domergue and B. Cormack
        pRD97 msn4ΔA 0.564-kb KpnI/XhoI PCR fragment (corresponding to primers 2990 and 2991) carrying the promoter region of MSN4 and a 0.535-kb BamHI/SacI PCR fragment (corresponding to primers 2992 and 2993) carrying the 3′ UTR of MSN4 were cloned into pAP599 AprR. Domergue and B. Cormack
  • TABLE 3.

    Oligonucleotides used in this study

    PrimerSequenceaDescription or restriction site(s)
    13 GTTGTAAAACGACGGCCAGTG pUC forward
    17 GGAAACAGCTATGACCATG pUC reverse
    1 CGCGGATCCAAGTATACTGCTATGAGCTAC BamHI
    2 CAAGGAGCTCTTGTGCAGCGTGTAAGATATGAATCAAGTGAT SacI/BsgI
    4 TCAGATCTCGAGTTGACCGTGACCGAAAC XhoI
    5 CCGGGTACCTTGTGCAGTCGAAGTTAGAGTGCCCTATTC KpnI/BsgI
    7 AATCTCGAGTTGTGCAGTGCGGGTAACAATTCTCGGCG XhoI/BsgI
    8 CCCAAGCTTTTACTTCCTAGTTCTTGTCTC HindIII
    11 CGCGGATCCTGTCTATATTATCTCGGTAGATC BamHI
    12 CAAGGAGCTCTTGTGCAGTCAACTCATAGATCACAACATTAACAC SacI/BsgI
    57 CGCGGATCCTCAATTGTGGGAAGTTATCTAATAAGCC BamHI
    58 CACTACTAGTTAAACACTTGTAGGAG SpeI
    60 CCCAAGCTTTTGAACCACGTAAAGTGCTGT HindIII
    61 CCGGGTACCTTGTGCAGCCTGTAAGGACTTCTAAACC KpnI/BsgI
    2984 GGTACCCGAGGGAGTTGCCTTCACTATGTGCGT TGAG KpnI/BcgI
    2985 CTCGAGCTGTTCTTGTTGATCTGTGTTTGGT XhoI
    2986 GGATTCTGACAGTGTTCCTTATTTTATCTAG BamHI
    2987 GAGCTCCGACCGCGGTGCATCTGTTACCAGGTTAGCC SacI/BcgI
    2990 GGTACCCGACTCCTGTGCCTTGTCGTACCAGAGAAAC KpnI/BcgI
    2991 CTCGAGGAACAGGAATGGACACTAATATATA XhoI
    2992 GGATCCCCATTCTTTATTTTATTTTCTGCTA BamHI
    2993 GAGCTCCGAGGCATCTGCTAATAATTTTCCGTTTCAA SacI/BcgI
    • ↵ a Restriction sites are indicated in boldface.

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High Resistance to Oxidative Stress in the Fungal Pathogen Candida glabrata Is Mediated by a Single Catalase, Cta1p, and Is Controlled by the Transcription Factors Yap1p, Skn7p, Msn2p, and Msn4p
Mayra Cuéllar-Cruz, Marcela Briones-Martin-del-Campo, Israel Cañas-Villamar, Javier Montalvo-Arredondo, Lina Riego-Ruiz, Irene Castaño, Alejandro De Las Peñas
Eukaryotic Cell May 2008, 7 (5) 814-825; DOI: 10.1128/EC.00011-08

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High Resistance to Oxidative Stress in the Fungal Pathogen Candida glabrata Is Mediated by a Single Catalase, Cta1p, and Is Controlled by the Transcription Factors Yap1p, Skn7p, Msn2p, and Msn4p
Mayra Cuéllar-Cruz, Marcela Briones-Martin-del-Campo, Israel Cañas-Villamar, Javier Montalvo-Arredondo, Lina Riego-Ruiz, Irene Castaño, Alejandro De Las Peñas
Eukaryotic Cell May 2008, 7 (5) 814-825; DOI: 10.1128/EC.00011-08
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    • ABSTRACT
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KEYWORDS

Candida glabrata
Candidiasis
Catalase
Oxidative Stress
Transcription Factors

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