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. 1996 Nov 12;93(23):13217-22.
doi: 10.1073/pnas.93.23.13217.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

E Leberer  1 , D Harcus, I D Broadbent, K L Clark, D Dignard, K Ziegelbauer, A Schmidt, N A Gow, A J Brown, D Y Thomas
Affiliations

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

E Leberer et al. Proc Natl Acad Sci U S A. .

Abstract

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

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Figures

Figure 1
Figure 1
The C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae diploid cells. Colonies of the diploid STE20 wild-type strain L5266 (4) (A) and the isogenic ste20/ste20 strain HLY492 (4) transformed with either the control plasmid pRS316 (B), the CST20 plasmid pRL53 (C), or the STE20 plasmid pSTE20-5 (15) (D) were grown on nitrogen starvation medium (2) for 5 days at 30°C. (×4; bar = 1 mm.)
Figure 2
Figure 2
Morphology of S. cerevisiae MATa cells (strain YEL306-1A) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 (A), STE20 (B) and C. albicans CST20 (C). The diploid strain YEL306 heterozygous for ste20Δ::TRP1/STE20 cla4Δ::LEU2/CLA4 (E.L., unpublished) was transformed with plasmid pRS316 carrying either no insert, CLA4 (pRL21), CST20 (pRL53), or STE20 (pSTE20-5), and then sporulated and dissected. No viable haploid ste20Δ cla4Δ spores were obtained from transformants with the plasmid without insert, but were obtained from transformants with plasmids carrying CLA4, STE20, or CST20. Cells were grown to mid-exponential phase in YPD medium at 30°C. No viable ste20Δ cla4Δ segregants were obtained in medium containing 5-fluoroorotic acid suggesting that the plasmids were essential for viability. Neither STE20 nor CST20 were able to suppress the morphological defect of cla4Δ cells. (×40; bar = 30 μm.)
Figure 4
Figure 4
(A) Deletion of CST20 in C. albicans. PCR with the divergent oligodeoxynucleotides ODH68 and ODH69 was used to partially delete the coding sequence of CST20. A hisG-URA3-hisG cassette was then inserted. (B) Southern blot analysis with a CST20 fragment from EcoRI–XbaI as a probe. The genomic DNA samples digested with XhoI were from following strains: Lanes: 1, CAI4 (ura3/ura3 CST20/CST20); 2, CDH15 (ura3/ura3 CST20/cst20Δ::hisG-URA3-hisG); 3, CDH18 (ura3/ura3 CST20/cst20Δ::hisG); 4, CDH22 (ura3/ura3 cst20Δ::hisG-URA3hisG/cst20Δ::hisG); and 5, CDH25 (ura3/ura3 cst20Δ::hisG/cst20Δ::hisG). (C) Deletion of HST7 in C. albicans. A HindIII–BglII fragment was replaced by the hisG-URA3-hisG cassette to create plasmid pPF-7. (D) Southern blot analysis was performed with the two large BamHI fragments of pPF-7 as probes recognizing the HST7 sequence upstream of the HindIII site and the hisG-URA3-hisG cassette. The genomic DNA samples digested with XbaI were from following strains. Lanes: 1, CAI4 (ura3/ura3 HST7/HST7); 2, CDH5 (ura3/ura3 HST7/hst7Δ::hisG-URA3-hisG); 3, CDH8 (ura3/ura3 HST7/hst7Δ::hisG); 4, CDH10 (ura3/ura3 hst7Δ::hisG-URA3-hisG/hst7Δ::hisG); and 5, CDH12 (ura3/ura3 hst7Δ::hisG/hst7Δ::hisG). The endonuclease restriction sites are as follows: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NsiI; P, PstI; Pv, PvuII; Sac, SacI; Sal, SalI; X, XbaI; Xh, XhoI.
Figure 5
Figure 5
Colonies of C. albicans cells grown for 5 days at 37°C on solid Spider medium containing mannitol. Wild-type strain SC5314 (13) (A), ura3/ura3 cst20Δ/cst20Δ::URA3 strain CDH22 (B), ura3/ura3 cst20Δ/cst20Δ::CST20::URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid pDH190) (C), ura3/ura3 cst20Δ/cst20Δ strain CDH25 transformed with plasmids pYPB1-ADHpt (D) and pYPB1-ADHpt-HST7 (E), ura3/ura3 hst7Δ/hst7Δ strain CDH12 transformed with plasmids pVEC (F), pVEC-HST7 (G), pYPB1-ADHpt (H), and pYPB1-ADHpt-HST7 (I), and ura3/ura3 cph1/cph1 strain CDH72 [ura3/ura3 derivative of strain JK19 (6)] transformed with pYPB1-ADHpt-HST7 (J). (×2; bar = 2 mm.)
Figure 6
Figure 6
Virulence assays. Survival curves of mice (n = 10 for each C. albicans strain at each inoculation dose) infected with 1 × 106 (A) and 1 × 105 (B) cells of C. albicans strains SC5314 (wild type), CAI4 (ura3/ura3), CDH22 (ura3/ura3 cst20Δ/cst20Δ::URA3), and CDH10 (ura3/ura3 hst7Δ/hst7Δ::URA3). (C) Staining of mouse kidney sections with periodic acid Schiff’s stain 48 h after infection with cst20Δ/cst20Δ::URA3 mutant strain CDH22 (a) and hst7Δ/hst7Δ::URA3 mutant strain CDH10 (b). Some hyphal cells are indicated with arrows. (Bar = 0.1 mm.)
Figure 6
Figure 6
Virulence assays. Survival curves of mice (n = 10 for each C. albicans strain at each inoculation dose) infected with 1 × 106 (A) and 1 × 105 (B) cells of C. albicans strains SC5314 (wild type), CAI4 (ura3/ura3), CDH22 (ura3/ura3 cst20Δ/cst20Δ::URA3), and CDH10 (ura3/ura3 hst7Δ/hst7Δ::URA3). (C) Staining of mouse kidney sections with periodic acid Schiff’s stain 48 h after infection with cst20Δ/cst20Δ::URA3 mutant strain CDH22 (a) and hst7Δ/hst7Δ::URA3 mutant strain CDH10 (b). Some hyphal cells are indicated with arrows. (Bar = 0.1 mm.)
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