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CELL GROWTH AND DEVELOPMENT

Ash1 Protein, an Asymmetrically Localized Transcriptional Regulator, Controls Filamentous Growth and Virulence of Candida albicans

Diane O. Inglis, Alexander D. Johnson
Diane O. Inglis
Department of Biochemistry and Biophysics and Department of Microbiology and Immunology, University of California—San Francisco, San Francisco, California 94143
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Alexander D. Johnson
Department of Biochemistry and Biophysics and Department of Microbiology and Immunology, University of California—San Francisco, San Francisco, California 94143
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  • For correspondence: ajohnson@cgl.ucsf.edu
DOI: 10.1128/MCB.22.24.8669-8680.2002
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  • FIG. 1.
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    FIG. 1.

    Sequence alignment of the predicted amino acid sequences from C. albicans and S. cerevisiae Ash1p. Boxes indicate identical residues. Three possible start codons for C. albicans Ash1p are indicated by dots. Two predicted serine residues encoded by nonstandard CUG codons of C. albicans (39) are indicated by asterisks. The highly conserved GATA-like zinc finger region is underlined. Amino acid numbers are indicated on the right.

  • FIG. 2.
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    FIG. 2.

    C. albicans Ash1p represses HO and promotes pseudohyphal growth in S. cerevisiae ash1 mutant strains. The HO-ADE2 HO-CAN1 ash1 reporter strain produces red colonies on 0.03% canavanine and 10 mg of adenine per ml of SD −Leu medium when transformed with a high-copy-number plasmid that carries either S. cerevisiae (A) or C. albicans (B) ASH1. The same strain transformed with vector (D) fails to grow on this medium; the few surviving colonies are white, indicating HO-ADE2 is not repressed. The HO-ADE2 HO-CAN1 ASH1 control strain (C) has the genomic copy of ASH1 intact and contains the vector control plasmid. Strains were incubated for 5 days at 30°C. For the experiments shown in panels E to G, diploid ash1/ash1 strains of the Σ1278b background were transformed with the indicated plasmids and then incubated on nitrogen-limiting SLAD medium for 5 days at 30°C. Colonies were photographed at a magnification of ×38.

  • FIG. 3.
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    FIG. 3.

    ASH1 is required for filamentous growth of C. albicans. Heterozygous or homozygous strains of the indicated genotypes were grown on various types of filament-inducing solid media: Spider medium (A), YPD medium plus 10% serum (B), and Lee's medium (pH 6.8) with 2% agar (C). Plates were incubated for 5 to 6 days at 30°C. In panel A, the strain depicted in the panel in the second row of the second column is an ash1/ash1 strain into which an intact copy of ASH1 has been introduced.

  • FIG. 4.
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    FIG. 4.

    Summary of the characteristics of the yeast- and hyphal-form cells examined in this study.

  • FIG. 5.
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    FIG. 5.

    Ash1p is localized to daughter cells of C. albicans growing in the yeast form. (A) Cells expressing myc-Ash1p (YDI-199) were grown in M199 (pH 4.5) at 23°C (conditions that favor the budding-yeast form) and processed for indirect immunofluorescence (see Materials and Methods). Cells were stained for myc-Ash1p with 9E10 mouse antibodies and Cy3-conjugated secondary antibodies that recognize the mouse 9E10 antibody. Tup1p was stained with rabbit polyclonal antibodies and FITC-conjugated secondary antibodies. Cell nuclei were visualized with DAPI stain, and whole cells were examined by bright-field imaging. Cells are stained as described in panel A. Yeast cells grown in M199 (pH 7.0) at 23°C appear as chains of attached budding cells. In panels A and B, selected mother (m) and daughter (d) cells are labeled.

  • FIG. 6.
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    FIG. 6.

    Ash1p localizes to hyphal tip cells. (A to D) Ash1p is observed in the hyphal daughter cell nucleus of mother-daughter hyphal cell pairs grown for 2 h at 35°C in YPD plus 20% serum (A, anti-myc stain; B, anti-Tup1p stain; C, DAP1 stain; D, bright-field image). (E to H) Hyphal cells grown for 4 to 6 h show chains of cells in a hyphal filament with Ash1p located only in the nucleus of the hyphal tip (apical) cell (indicated by the arrow). The original mother cell (m) has also produced budding cells. The images are as described for panels A to D.

  • FIG. 7.
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    FIG. 7.

    Mature hyphae produce budding daughter cells that express Ash1p. Cells were grown overnight at 35°C in YPD plus 20% serum and stained as described in the legend to Fig. 6.

  • FIG. 8.
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    FIG. 8.

    ASH1 is important for virulence in a mouse model of systemic candidiasis. Shown are survival curves of mice systemically infected with URA3+ C. albicans strains of the genotypes ASH1/ASH1 (CAF2-1), ASH1/ash1 (YDI-1), ash1/ash1 (YDI-7), and ash1/ash1::ASH1 (YDI-157) (A) or ASH1/ASH1 (CAF2-1), ash1/ash1 (YDI-27), ash1/ash1 cph1/cph1 (YDI-129), and cph1/cph1 (JKC19) (B).

  • FIG. 9.
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    FIG. 9.

    ash1/ash1 mutants colonize kidneys much more slowly than do ash1/ash1/ASH1+ reintegrants, although they do produce hyphae in vivo. Histological sections of mouse kidneys were prepared as described in Materials and Methods. (A, B) The ash1/ash1/ASH1+ strain 24 h after infection. Panel A shows a locus of infection on the edge of the kidney and a large colony inside the kidney (B). (C to F) Shown are the ash1/ash1 mutant strains after 2 (C and D), 15 (E), and 30 (F) days. Panels A to D were photographed at ×40, and panels E and F were photographed at ×20.

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  • TABLE 1.

    Strains used in this study

    StrainGenotypeSource or reference
    C. albicans a
        CAF2-1 URA3/ura3::imm434 13
        CAI4 ura3Δ/ura3Δ 13
        RM1000 ura3Δ/ura3Δ his1::HisG/his1::HisG 34
        YDI-1 ura3Δ/ura3Δ ASH1/ash1::HisG-URA3-HisG This study
        YDI-7 ura3Δ/ura3Δ ash1::HisG/ash1::HisG-URA3-HisG This study
        YDI-11 ura3Δ/ura3Δ ash1::HisG/ash1::HisG This study
        YDI-129 ura3Δ/ura3Δ cph1::HisG/cph1::HisG ash1::HisG/ash1::HisG-URA3-HisG This study
        YDI-154 ura3Δ/ura3Δ ash1::HisG/ash1::HisG::ASH1-URA3 (2d) This study
        YDI-157 ura3Δ/ura3Δ ash1::HisG/ash1::HisG::ASH1-URA3 (6d) This study
        YDI-199 ura3Δ/ura3Δ ash1::HisG/ash1::HisG-URA3-HisG::myc6-ASH1 This study
        JKC19 ura3Δ/ura3Δ cph1::HisG/cph1::HisG-URA3-HisG 29
    S. cerevisiae b
        CGX69 MAT a/MATα ura3/ura3 17
        YDI-56 MAT a/MATα ura3/ura3 ash1::HisG/ash1::HisG This study
        YAS242-10B MATα ASH1 can1::HO-CAN1 ho::HO-ADE2 ade2-1 his3-11,15 trp1-1 ura3 leu2-3,112 Sil, unpublished
        YAS234-1A MATα ash1::TRP1 can1::HO-CAN1 ho::HO-ADE2 ade2-1 his3-11,15 trp1-1 ura3 leu2-3,112 Sil, unpublished
    • ↵ a All C. albicans strains listed as ura3Δ/ura3Δ are homozygous for the ura3::lmm434 mutation, except for CAF2-1, which is heterozygous for the mutation.

    • ↵ b CGX69 and YDI-56 are of the Σ1278b strain background; YAS242-10B and YAS243-1A are of the W303 strain background.

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Ash1 Protein, an Asymmetrically Localized Transcriptional Regulator, Controls Filamentous Growth and Virulence of Candida albicans
Diane O. Inglis, Alexander D. Johnson
Molecular and Cellular Biology Dec 2002, 22 (24) 8669-8680; DOI: 10.1128/MCB.22.24.8669-8680.2002

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Ash1 Protein, an Asymmetrically Localized Transcriptional Regulator, Controls Filamentous Growth and Virulence of Candida albicans
Diane O. Inglis, Alexander D. Johnson
Molecular and Cellular Biology Dec 2002, 22 (24) 8669-8680; DOI: 10.1128/MCB.22.24.8669-8680.2002
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KEYWORDS

Candida albicans
transcription factors
Transcription, Genetic

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