Crk1, a novel Cdc2-related protein kinase, is required for hyphal development and virulence in Candida albicans

Abstract

Both mitogen-activated protein kinases and cyclin-dependent kinases play a role in hyphal development in Candida albicans. Using an oligonucleotide probe-based screen, we have isolated a new member of the Cdc2 kinase subfamily, designated Crk1 (Cdc2-related kinase). The protein sequence of Crk1 is most similar to those of Saccharomyces cerevisiae Sgv1 and human Pkl1/Cdk9. In S. cerevisiae, CRK1 suppresses some, but not all, of the defects associated with an sgv1 mutant. Deleting both copies of CRK1 in C. albicans slows growth slightly but leads to a profound defect in hyphal development under all conditions examined. crk1/crk1 mutants are impaired in the induction of hypha-specific genes and are avirulent in mice. Consistent with this, ectopic expression of the Crk1 kinase domain (CRK1N) promotes filamentous or invasive growth in S. cerevisiae and hyphal development in C. albicans. The activity of Crk1 in S. cerevisiae requires Flo8 but is independent of Ste12 and Phd1. Similarly, Crk1 promotes filamentation through a route independent of Cph1 and Efg1 in C. albicans. RAS1(V13) can also activate filamentation in a cph1/cph1 efg1/efg1 double mutant. Interestingly, CRK1N produces florid hyphae in ras1/ras1 strains, while RAS1(V13) generates feeble hyphae in crk1/crk1 strains.

FIG. 1

Comparison of the Crk1 sequence to sequences of other Cdc2-related kinases. (A) Diagram of predicted functional domains in Crk1. The shaded region contains the conserved kinase domain, as shown in panel B. Potential nuclear localization sequences (based on the PSORT program) near the carboxyl terminus are also indicated. (B) Sequence alignment of C. albicans Crk1 (CaCrk1) kinase domains with those of S. cerevisiae Sgv1 (ScSgv1), an S. pombe Cdc2 homologue (SpCdc2h; accession no. AB004534), a human Ser/Thr kinase (hukinase; accession no. AB020711), the human PITALRE kinase HuKp11/Cdk9, S. cerevisiae Cdc28, and S. cerevisiae Fus3. Subdomains are labeled according to Hanks et al. (27). Shaded residues represent identities among these kinases. Conserved phosphorylation sites in Cdc28 and Fus3 are indicated with asterisks and dots, respectively. Underlined sequences denote the insertion unique for the Cdc2 branch of the kinases. The arrow indicates the ending position of Crk1N and Sgv1N. (C) Kinase activity associated with Crk1 and Crk1N immunocomplexes. Yeast cell extracts were immunoprecipitated with anti-HA antibodies. Immunocomplexes were assayed for the ability to phosphorylate MBP (2-h exposure).

FIG. 2

Suppression of S. cerevisiae sgv1 mutants by C. albicans Crk1. (A) Pheromone-induced growth arrest assay. Haploid S. cerevisiae sgv1 mutants were transformed with vector (pVTU) (1), SGV1 (pVTUSGV1) (2), SGV1N (pVTUSGV1N) (3), CRK1 (pVTUCRK1) (4), and CRK1N (pVYUCRK1N) (5). Approximately 10⁷ cells were plated on each YPD plate. Sterile filter disks were placed on the nascent cell lawns; α-factor in the amounts of 0 ng (top), 50 ng (left), and 500 ng (right) was added to the disks. Plates were incubated for 2 days at 30°C. (B) Effect of temperature on growth. The strains used for panel B were used to test growth properties at 37 and 18°C. Cells were streaked onto SC−Ura plates, which were incubated for 5 days at 30°C, 7 days at 37°C, and 7 days at 18°C.

FIG. 3

Disruption of the C. albicans CRK1 gene. (A) Restriction map and disruption strategy for CRK1. (B) Southern analysis of transformants with the CRK1 disruption construct. Genomic DNA from the recipient strain (lane 1, CAI4), a heterozygote transformant (lane 2, CAW1), an FOA^r/ura3 derivative of CAW1 (lane 3, CAW2), a homozygote transformant (lane 4, CAW3), and an FOA^r/ura3 derivative of CAW3 (lane 5, CAW4) were digested with BamHI. The Southern blot on the left was probed with the 4-kb SacI fragment shown in panel A. The BamHI site in the hisG-URA3-hisG sequence generated two new hybridization fragments of 10.4 and 1.4 kb from the original 9-kb wild-type BamHI fragment. The 10.4-kb crk1::hisG-URA3-hisG fragment became an 8-kb crk1::hisG fragment after selection on an FOA plate to loop out the URA3 and one copy of hisG. This size difference between crk1::hisG and crk1::hisG-URA3-hisG is evident in lane 4, where the doublet represents fragments of 10.4 and 8 kb, respectively. The Southern blot in the middle was probed with the 2-kb EcoRV-XhoI fragment shown in panel A. The EcoRV-XhoI region was replaced with the hisG-URA3-hisG sequence in the deletion construct. Therefore, the 2-kb probe is expected to hybridize only to the 9-kb BamHI fragment from the wild-type CRK1 locus. Homozygous crk1/crk1 mutants do not contain the 9-kb BamHI fragment. The Southern blot on the right was probed with C. albicans URA3.

FIG. 4

Effects of CRK1 disruption on cell growth. (A) crk1/crk1 strains grow slower than wild type. Wild-type (WT; SC5314), CRK1/crk1 (CAW1), crk1/crk1 (CAW3), crk1/crk1 carrying a vector (CAW5), and crk1/crk1 carrying CRK1 (CAW6) were grown on a YPD plate for 5 days at 22°C. (B) Comparison of cell morphologies. Wild-type (SC5314) and crk1/crk1 (CAW3) cells were grown in YPD medium at 22°C for 15 h and photographed.

FIG. 5

crk1/crk1 strains are defective in hyphal development. (A) crk1/crk1 strains cannot develop hyphal colonies. Ura⁺ strains, including wild type (WT; SC5314), CRK1/crk1 (CAW1), crk1/crk1 (CAW3), and crk1/crk1 carrying ectopically expressed CRK1 (CAW6), were plated at a density of about 50 colonies per plate on solid serum-containing medium and solid Lee's medium. Colonies were photographed after incubation at 37°C for the days (d) indicated. (B) crk1/crk1 strains are impaired in hyphal filament formation in liquid media. Overnight cultures of the four Ura⁺ strains used for panel A were diluted in YPD medium containing 10% serum or modified Lee's medium for hyphal induction. Cells were photographed after incubation at 37°C for the time indicated. (C) crk1/crk1 mutants are defective in induction of hypha-specific genes. RNA from wild-type cells grown in YPD at 30°C for 6 h was used as a control for gene expression in the yeast growth form (left). RNA from cells of the same Ura⁺ strains induced for 3.5 h by serum (panel B, top row) and induced for 6 h in Lee's medium (panel B, middle row) were subjected to Northern analysis, as shown in panel C, middle and right gels, respectively. Northern probes are as indicated. The image for the CRK1 Northern blot was obtained after 3 days of exposure. Images for ECE1-, HWP1-, and ACT1-probed filters were obtained after 3 h of exposure. Transcript levels were quantified with a PhosphorImager.

FIG. 5

crk1/crk1 strains are defective in hyphal development. (A) crk1/crk1 strains cannot develop hyphal colonies. Ura⁺ strains, including wild type (WT; SC5314), CRK1/crk1 (CAW1), crk1/crk1 (CAW3), and crk1/crk1 carrying ectopically expressed CRK1 (CAW6), were plated at a density of about 50 colonies per plate on solid serum-containing medium and solid Lee's medium. Colonies were photographed after incubation at 37°C for the days (d) indicated. (B) crk1/crk1 strains are impaired in hyphal filament formation in liquid media. Overnight cultures of the four Ura⁺ strains used for panel A were diluted in YPD medium containing 10% serum or modified Lee's medium for hyphal induction. Cells were photographed after incubation at 37°C for the time indicated. (C) crk1/crk1 mutants are defective in induction of hypha-specific genes. RNA from wild-type cells grown in YPD at 30°C for 6 h was used as a control for gene expression in the yeast growth form (left). RNA from cells of the same Ura⁺ strains induced for 3.5 h by serum (panel B, top row) and induced for 6 h in Lee's medium (panel B, middle row) were subjected to Northern analysis, as shown in panel C, middle and right gels, respectively. Northern probes are as indicated. The image for the CRK1 Northern blot was obtained after 3 days of exposure. Images for ECE1-, HWP1-, and ACT1-probed filters were obtained after 3 h of exposure. Transcript levels were quantified with a PhosphorImager.

FIG. 5

crk1/crk1 strains are defective in hyphal development. (A) crk1/crk1 strains cannot develop hyphal colonies. Ura⁺ strains, including wild type (WT; SC5314), CRK1/crk1 (CAW1), crk1/crk1 (CAW3), and crk1/crk1 carrying ectopically expressed CRK1 (CAW6), were plated at a density of about 50 colonies per plate on solid serum-containing medium and solid Lee's medium. Colonies were photographed after incubation at 37°C for the days (d) indicated. (B) crk1/crk1 strains are impaired in hyphal filament formation in liquid media. Overnight cultures of the four Ura⁺ strains used for panel A were diluted in YPD medium containing 10% serum or modified Lee's medium for hyphal induction. Cells were photographed after incubation at 37°C for the time indicated. (C) crk1/crk1 mutants are defective in induction of hypha-specific genes. RNA from wild-type cells grown in YPD at 30°C for 6 h was used as a control for gene expression in the yeast growth form (left). RNA from cells of the same Ura⁺ strains induced for 3.5 h by serum (panel B, top row) and induced for 6 h in Lee's medium (panel B, middle row) were subjected to Northern analysis, as shown in panel C, middle and right gels, respectively. Northern probes are as indicated. The image for the CRK1 Northern blot was obtained after 3 days of exposure. Images for ECE1-, HWP1-, and ACT1-probed filters were obtained after 3 h of exposure. Transcript levels were quantified with a PhosphorImager.

FIG. 6

Virulence assay. ICR male mice were injected with wild-type (WT; SC5314; A), CRK1/crk1 (CAW1; B), crk1/crk1 (CAW3; C), hst7/hst7 (JKC129; D), cph1/cph1 (JKC19; E), and cph1/cph1 efg1/efg1 (HLC54; F) strains. The mice were injected with 5 × 10⁵ (●) and 5 × 10⁶ (×) C. albicans cells. Mice injected with either crk1/crk1 cells or cph1/cph1 efg1/efg1 cells all survived for more than 20 days.

FIG. 7

CRK1N stimulated filamentous and invasive growth in S. cerevisiae. (A) Colony morphologies of isogenic wild-type (WT; CG31), ste7/ste7 (HLY351), ste12/ste12 (HLY352), tec1/tec1 (HLY2002), phd1/phd1 ste12/ste12 (L6235), and flo8/flo8 (HLY852) strains carrying vector (left) or CRK1N (right) grown on SLAD at 30°C for 4 days. (B) Total and invasive growth of wild-type (L5528), ste7 (HLY367), ste12 (HLY362), tec1 (HLY2000), and flo8 (HLY850) strains carrying a vector (left) or CRK1N (right) after 5 days of growth on SC−Ura.

FIG. 8

Ectopic expression of CRK1- or CRK1N-promoted filamentation under yeast growth conditions. (A) Colony phenotypes of wild-type (SC5314), crk1/crk1 with vector (CAW5), crk1/crk1 with ectopic expression of CRK1 (CAW6), and CRK1N (CAW7) strains grown on YPD medium at 30°C for 3 days. (B) Induction of ECE1 transcription by ectopic expression of CRK1 and CRK1N. Wild-type (SC5314), CRK1/crk1 (CAW1), crk1/crk1 carrying a vector (V; CAW5), crk1/crk1 carrying CRK1 (CAW6), and crk1/crk1 carrying CRK1N (CAW7) strains were grown in YPD at 30°C for 6 h, and total RNA was extracted for Northern analysis. The Northern blot was probed with CRK1, ECE1, and ACT1 and exposed for 3 days (for the CRK1 and ECE1 transcripts) and 3 h (for ACT1).

FIG. 9

Functional relationship of Crk1 with the filamentation MAP kinase pathway, Efg1, and Ras1 in C. albicans hyphal development. The C. albicans mutant strains indicated on the left (described in Table 1) were transformed with CRK1N (BES119CRK1N) and RAS1^V13 (pQF145.2). Both genes are under the control of the MAL2 promoter. The C. albicans transformants were grown on an SC−Ura+sucrose (2%) plate containing 50 mM succinate at pH 5 for 4 days at 30°C (20). Well-separated colonies near the edge of each steak were photographed.