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

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.

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.

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

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.

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.

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.

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).