Protein phosphatase Pph3 and its regulatory subunit Psy2 regulate Rad53 dephosphorylation and cell morphogenesis during recovery from DNA damage in Candida albicans

Abstract

The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3Δ and psy2Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation.

Fig. 1.

Sequence and domain comparison of CaPph3 and CaPsy2 with S. cerevisiae orthologues. (A) Alignment of the CaPph3 amino acid sequence with that of ScPph3. Dark shading indicates identical residues, and light shading indicates similar residues. (B and C) Domain organizations of CaPph3 (B) and CaPsy2 (C) compared with their S. cerevisiae counterparts.

Fig. 2.

Effects of PPH3 and PSY2 deletion on growth rate and cell morphology. (A) Cells (5 × 10⁵) of wild-type (WT) (SC5314, BWP17, and BWP17 ura⁺ strains gave similar results in the experiments described in this study), pph3Δ (SJL3), and psy2Δ (SJL6) strains (all strains used are listed in Table 1) were inoculated into liquid YPD medium in starting cultures. The cells were counted microscopically every 15 min. The experiment was done in triplicate, and the average of each time point was used to generate the growth curves. The error bars indicate 1 standard deviation (SD). (B) Cell morphology. Cells from the same strains as in panel A were grown in YPD medium at 30°C for 16 h or in YPD medium containing 20% serum at 37°C for 3 h. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (C) pph3Δ (SJL3) and psy2Δ (SJL6) mutants showed decreased filamentation under embedded conditions. Cells were embedded in YPD agar and grown at 30°C for 48 h.

Fig. 3.

pph3Δ and psy2Δ mutant cells are hypersensitive to DNA-damaging agents. Cells of WT (SC5314 or BWP17), pph3Δ (SJL3), and psy2Δ (SJL6) strains and the rescued PPH3 (SJL2.1) and PSY2 (SJL5.1) strains were serially diluted, spotted onto YPD plates containing different concentrations of cisplatin (CP) and MMS, and incubated at 30°C for 24 h. For UV treatment, serially diluted cells were spotted onto YPD plates and then irradiated with UV (80 or 160 J/m²), followed by incubation at 30°C for 24 h. All experiments were repeated using at least 3 independent clones for each mutant.

Fig. 4.

pph3Δ and psy2Δ cells exhibit irreversible pseudohyphal growth and cell cycle arrest when treated with MMS. (A) WT (SC5314 or BWP17), pph3Δ (SJL3), and psy2Δ (SJL6) yeast cells were grown in YPD medium supplemented with 0.02% MMS at 30°C. Cells were collected for microscopic examination at timed intervals. Bar = 5 μm. (B) Comparison of filament lengths of cells treated with MMS. Filament length was measured using ImageJ (http://rsbweb.nih.gov/ij/index.html). The data points show the average of 30 cells. The experiment was repeated 3 times. The error bars indicate 1 SD. (C) Cells were treated as described for panel A and harvested at intervals for flow cytometry analysis. (D and E) The same cells used in panel A were incubated in YPD medium containing 0.02% MMS for 6 h before washing the cells with fresh YPD and inoculating the MMS-treated cells into MMS-free YPD medium. Aliquots were harvested at intervals for microscopy and flow cytometry analysis. (F) pph3Δ and psy2Δ cells invade agar during recovery from DNA damage. BWP17 and mutant (SJL3 and SJL6) cells that had been grown in YPD medium containing 0.02% MMS for 6 h were spread onto YPD agar plates and incubated at 30°C. As ∼90% of the mutant cells lost viability, after the MMS treatment, the cells were first concentrated by ∼10-fold before being spread onto the plates so that more colonies would grow. The plates were photographed after 3 days and then washed to remove cells on the agar surface and rephotographed.

Fig. 5.

PPH3 and PSY2 are dispensable for CaHta dephosphorylation during recovery from genotoxic stress. (A) BWP17 cells were incubated with various concentrations of MMS or cisplatin for 1 h and then harvested for immunoblot analysis using the anti-Hta-P antibody. Cdc28 was probed with anti-PSTAIRE antibody as a loading control. (B and C) Wild-type (BWP17), pph3Δ (SJL3), and psy2Δ (SJL6) cells were incubated in YPD medium containing 0.02% MMS or 2 mM cisplatin for 2 h. MMS and cisplatin were then washed out, and the cells were resuspended in YPD medium and incubated at 30°C. Cells were harvested at the indicated time points for immunoblot analysis using the Hta-P antibody. Cdc28 was probed with anti-PSTAIRE antibody as a loading control.

Fig. 6.

Pph3 and Psy2 regulate the phosphorylation state of Rad53 in C. albicans. (A) Wild-type (SJL9), pph3Δ (SJL7), and psy2Δ (SJL8) cells were incubated in YPD medium containing 0.02% MMS. Cells were harvested at the indicated time points for immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as a loading control. (B) Rad53 hyperphosphorylation in MMS-treated cells. The lysate of SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy3Δ RAD53-Myc) cells that had been grown in the presence of (+) 0.02% MMS for 4 h was divided into 2 parts. One was treated with λ-phosphatase (PPase), and the other was mock treated with the reaction buffer alone. The two samples, along with untreated cell lysates, were then subjected to Western blot analysis using anti-Myc antibody. (C) Wild-type (SJL9), pph3Δ (SJL7), and psy2Δ (SJL8) cells were incubated in YPD medium containing 0.02% MMS or 2 mM cisplatin for 6 h. Untreated cells (−) were included as a control. MMS and cisplatin were washed out, and the cells were resuspended in YPD medium at 30°C. The cells were harvested at the indicated time points for immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as a loading control.