Linking Sfl1 Regulation of Hyphal Development to Stress Response Kinases in Candida albicans

Abstract

Candida albicans is an important human pathogen responsible for causing both superficial and systemic infections. Its ability to switch from the yeast form to the hyphal growth form is required for its pathogenicity. Acidic pH inhibits hyphal initiation, but the nature of the mechanism for this inhibition is not completely clear. We show that acidic pH represses hyphal initiation independently of the temperature- and farnesol-mediated Nrg1 downregulation. Using a collection of transcription factor deletion mutants, we observed that the sfl1 mutant induced hyphae in acidic pH but not in farnesol at 37°C. Furthermore, transcription of hyphal regulators BRG1 and UME6 was not induced in wild-type (WT) cells but was induced in the sfl1 mutant during hyphal induction in acidic pH. Using the same screening conditions with the collection of kinase mutants, we found that deletions of the core stress response mitogen-activated protein (MAP) kinase HOG1 and its kinase PBS2, the cell wall stress MAP kinase MKC1, and the calcium/calmodulin-dependent kinase CMK1 allowed hyphal initiation in acidic pH. Furthermore, Hog1 phosphorylation induced by high osmotic stress also retarded hyphal initiation, and the effect was abolished in the sfl1 and three kinase mutants but was enhanced in the phosphatase mutant ptp2 ptp3 We also found functional associations among Cmk1, Hog1, and Sfl1 for cation stress. Our study results suggest that robust hyphal initiation requires downregulation of both Nrg1 and Sfl1 transcriptional repressors as well as timely BRG1 expression. Acidic pH and cationic stress retard hyphal initiation via the stress-responsive kinases and Sfl1.IMPORTANCECandida albicans is a commensal as well as a pathogen of humans. C. albicans is able to mount a cellular response to a diverse range of external stimuli in the host and switch reversibly between the yeast and hyphal growth forms. Hyphal development is a key virulence determinant. Here, we studied how C. albicans senses different environmental signals to control its growth forms. Our study results suggest that robust hyphal development requires downregulation of two transcriptional repressors, Nrg1 and Sfl1. Acidic pH or cationic stress inhibits hyphal formation via stress-responsive kinases and Sfl1.

Keywords: Candida albicans; Sfl1; hyphal formation.

FIG 1

(A) Promoter shutdown assay to compare the levels of Nrg1 stability at pH 4 and pH 7 with those of the WT strain containing a copy of MAL2p-NRG1-MYC. A parallel blot was probed with anti-PSTAIRE antibody as a loading control. (B) Reverse transcription-quantitative PCR (qRT-PCR) of NRG1 transcript level after WT cells were grown at pH 4 and pH 7 for 1 h. Quantitative PCR (qPCR) values were normalized to ACT1 values for each sample, and overnight (ON) samples were set to a value of 1. Presented data represent means ± standard errors of the means (SEM) of results from 3 independent experiments. (C) Western blot analysis of Nrg1-myc protein levels after WT cells were inoculated into fresh medium at pH 4 and pH 7 for 1 h. ON, overnight. (D) Chromatin immunoprecipitation (ChIP) analysis of Nrg1 for the promoter of HWP1 after 30 min in YPD medium at pH 4 and pH 7. Presented data represent means ± SEM of results from 3 independent experiments.

FIG 2

(A) Morphology of WT and sfl1 strains expressing a copy of HWP1p-GFP after inoculation for 1 h in YPD medium set at pH 7 or pH 4 or supplemented with 100 μM farnesol. Percent filamentation is indicated at bottom right of DIC images. (B) qRT-PCR of BRG1 and UME6 transcripts after WT and sfl1 cells were grown at pH 4 and pH 7 for 1 h. qPCR values were normalized to CDC28 transcript levels for each sample. Presented data represent means ± SEM of results from 3 independent experiments. (C) Morphology of WT strain expressing a copy of MAL2p-BRG1 after inoculation for up to 2 h in YEP medium at pH 4 with either dextrose (+Dex) or maltose (+Mal) as the carbon source.

FIG 3

Morphology of WT, sfl1, hog1, cmk1, and mkc1 strains expressing a copy of HWP1p-GFP after inoculation for 1 h in YPD medium set at pH 7 or pH 4. Filamentation percentages are indicated at bottom right of DIC images.

FIG 4

(A) Acidic pH does not induce Hog1 phosphorylation. A Phospho-Hog1 immunoblot of cells grown for 3 h to the logarithmic phase and inoculated into fresh YPD medium at pH 4, pH 7, or pH 7 with 0.5 M NaCl for 5 min is shown. A pbs2 mutant strain was induced in 0.5 M NaCl as a negative control. A parallel blot was probed with anti-PSTAIRE as a loading control. (B) Acidic pH sustains Hog1 basal phosphorylation. A Phospho-Hog1 immunoblot of overnight cells (0 h) inoculated into fresh YPD medium at pH 4 and pH 7 for 3 h is shown. Aliquots were collected every hour. At 3 h, an aliquot of cells were shifted to medium with 1 M NaCl (3N) to induce Hog1 phosphorylation. A parallel blot was probed with anti-PSTAIRE as a loading control. (C) Acidic pH inhibits PTP3 transcription. qRT-PCR of WT and pbs2 cells was performed to measure the levels of PTP3 transcript after cells were grown at pH 4 and pH 6 for 15 min. qPCR values were normalized to ACT1 for each samples, and overnight (ON) samples were set to a value of 1. (D) Hog1 phosphorylation inhibits hyphal initiation. Morphology and HWP1p-GFP expression of WT, ptp2 ptp3, and ptp2 ptp3 hog1 strains expressing a copy of HWP1p-GFP after inoculation for 2.5 h in YPD medium at pH 7 or pH 4 or supplemented with 0.5 M NaCl are shown.

FIG 5

Morphology and GFP expression of WT, sfl1, hog1, cmk1, and mkc1 strains expressing a copy of HWP1p-GFP inoculated for 1 h and 2.5 h in YPD medium supplemented with 0.5 M NaCl. Percent filamentation is indicated on bottom right of DIC images.

FIG 6

Cation stress sensitivity of the WT, sfl1, hog1, cmk1, and mkc1 strains on YPD plates containing 0.3 M LiCl or 0.5 M NaCl.

FIG 7

Model of signal integration during hyphal initiation.