Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis

Abstract

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

FIG. 1

(A) Deletion of CEK1 in C. albicans; restriction map of and deletion strategy for the CEK1 gene. PCR with the divergent oligodeoxynucleotides OT1 and OT2 was used to delete a 1.2-kb fragment of the CEK1 gene. A 4.0-kb hisG-URA3-hisG cassette was then inserted. Restriction sites are as follows: B, BamHI; Bg, BglII; P, PstI; S, SacI; N, NsiI; E, EcoRI; Sp, SpeI. (B) Southern blot analysis of CEK1 disruptions with a 3.2-kb KpnI-SacI fragment containing the CEK1 gene as a probe. Genomic DNA samples from the following strains were digested with SpeI (absent from the hisG-URA3-hisG cassette): CEK1/CEK1 (CAI4) (lane 1), CEK1/cek1Δ::hisG-URA3-hisG (CK43A) (lane 2), CEK1/cek1Δ::hisG (CK43AL) (lane 3), cek1Δ::hisG-URA3-hisG/cek1Δ::hisG (CK43B-16) (lane 4), and cek1Δ::hisG/cek1Δ::hisG (CK43B-16L) (lane 5). Strains CK43B-4 and CK43B-4L gave results identical to the example shown here. Hybridization of a very small part (between the SacI and SpeI sites) of the probe to a 1.4-kb SpeI fragment, present only in the wild-type CEK1 gene, was barely detectable in these Southern blots and was not used for diagnostic purposes. The figure was assembled with Adobe Photoshop 3.0. Numbers on the left indicate sizes in kilobases.

FIG. 2

Growth of heterozygous and homozygous CEK1 deletion mutants on medium containing mannitol as a carbon source. Shown are uracil prototrophic strains: SC5314 (parental strain), CEK1/cek1Δ (CK43A), cek1Δ/cek1Δ (CK43B-16, identical to CK43B-4), cek1Δ/cek1Δ pVEC (plasmid vector pVEC in CK43B-4L), and cek1Δ/cek1Δ pVEC-CEK1 (plasmid pCCa4 containing the CEK1 gene in CK43B-4L). Cells were grown for 4 days at 30°C. Lee M medium (shown) and Spider medium gave similar results. Bar = 1.5 mm. The figure was assembled with Adobe Photoshop 4.0.

FIG. 3

Defective formation of parallel-sided hyphae from uracil prototrophic MAPK cascade null mutants grown on low-ammonia–dextrose medium (SLAD). Shown are SC5314 (wild-type parent), cst20Δ/cst20Δ (CDH22), hst7Δ/hst7Δ (CDH9), cek1Δ/cek1Δ (CK43B-16), cph1Δ/cph1Δ (JKC19), and cpp1Δ/cpp1Δ (CP29-1-7). Cells were grown for 7 days at 30°C. (2× objective, bar = 1.5 mm; 40× objective, bar = 90 μm.) Constrictions around septa were examined. Parallel-sided hyphae with no constrictions at septa are seen emanating from the borders of SC5314 and cpp1Δ/cpp1Δ colonies. Pseudohyphal filaments of cst20Δ/cst20Δ, hst7Δ/hst7Δ, cek1Δ/cek1Δ, and cph1Δ/cph1Δ strains are aberrant in shape and have constrictions between cells. Noninvasive cells from colonies scraped off plates were composed entirely of blastospores (data not shown). The figure was assembled with Adobe Photoshop 4.0.

FIG. 4

Complementation analysis of null mutants grown in SLAD medium. Shown are the uracil auxotrophic strains CAI4 (parent), cst20Δ/cst20Δ (CDH25), hst7Δ/hst7Δ (CDH12), cek1Δ/cek1Δ (CK43B-16L), and cph1Δ/cph1Δ (CDH72) transformed with pADH (YPB1-ADHpl), pADH-HST7 (pYPB1-ADHpt-HST7), or pADH-CPH1 (pLJ19). Cells were grown for 10 days at 30°C. As described above, filaments were examined for constrictions and for the radial penetration of filamentous growth beyond colony borders. The zones of filamentous growth surrounding pADH vector-transformed cst20/cst20 null mutants were composed of constricted pseudohyphae (as seen above), as were the less extensive filaments emanating from pADH vector-transformed hst7Δ/hst7Δ, cek1Δ/cek1Δ, and cph1Δ/cph1Δ null mutants. Bar = 1.5 mm. The figure was assembled with Adobe Photoshop 4.0.

FIG. 5

Growth of invasive mycelial colonies on solidified serum. (A) Cells grown for 24 h on serum at 37°C (magnification, ×20; bar = 0.75 mm). Shown are uracil prototrophic strains SC5314 (parental strain) (a), cek1Δ/cek1Δ (CK43B-4) (b), and cek1Δ/cek1Δ (CK43B-4L) (c) transformed with the pVEC-CEK1 plasmid pCCa4. (B) Cells grown for 3 days on serum at 37°C (magnification, ×2; bar = 3 mm). Shown are uracil prototrophic strains SC5314 (parental strain) (a), CEK1/cek1Δ (CK43A) (b), CEK1/cek1Δ::CEK1-URA3 (CK43A-RI) (c), cek1Δ/cek1Δ (CK43B-4) (d), cek1Δ/cek1Δ cpp1Δ/cpp1Δ (CP29-1-7CK14) (e), and cpp1Δ/cpp1Δ (CP29-1-7) (f). The figure was assembled with Adobe Photoshop 4.0. Panels A and B are from independent experiments.

FIG. 6

Survival curves for BALB/c mice injected intravenously with 5 × 10⁵ cells of the uracil prototrophic strains SC5314 (n = 5 mice; clinical isolate), cek1Δ/cek1Δ (CK43B-4; n = 8 mice), and cek1Δ/cek1Δ pVEC-CEK1 (CK43B-4L transformed with the plasmid pCCa4; n = 8 mice). Results were confirmed in two independent experiments. The cek1 null mutant strain CK43B-16 and another independent null mutant gave similar results (data not shown). Mortality data were quantified on days of sampling.

FIG. 7

Model of the C. albicans MAPK cascade for the yeast-to-hypha transition. CaSte11p is an undescribed putative homolog of the S. cerevisiae Ste11p MAPKKK whose role has not yet been examined for this pathway. TF, transcription factor. The phosphorylated form of Cek1p is designated by an asterisk.