Surface stress induces a conserved cell wall stress response in the pathogenic fungus Candida albicans

Abstract

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using (15)N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.

Fig 1

Spot assays of Candida albicans grown at 37°C and 42°C in the presence and absence of chemical stressors. SC5314 overnight cultures in 1:10 serial dilutions were spotted onto YNB-S plates at pH 4 or pH 7.4 and supplemented with 100 mg/liter SDS, 50 mg/liter calcofluor white, or 1 mg/liter Congo red, respectively, and grown for 2 days.

Fig 2

Relative quantification of wall proteins using FT-MS and ¹⁵N labeling. The graphs represent log₂ ratios of wall proteins at 42°C versus 37°C, at both pH 4 (median coefficient of variation, 13.1%) and pH 7.4 (median coefficient of variation, 11.2%). Positive values represent an increase and negative values represent a decrease of the corresponding protein at 42°C compared to the 37°C control.

Fig 3

Immunoblot analysis of phosphorylated MAP kinases. The cultures were grown for 18 h at pH 7.4 and 37°C in the presence of various chemical stressors (0.5 mg/liter fluconazole, 125 mg/liter SDS, 5 mg/liter Congo red, and 40 mg/liter calcofluor white) or subjected to thermal stress at 42°C. Fifteen micrograms of protein extracts was loaded per lane. Pgk1 was used as a loading control.

Fig 4

Temperature, stress conditions, and chitinase treatment influence the morphology of C. albicans wild-type and mutant strains. C. albicans wild-type and mutants were grown in YNB-S medium at pH 7.4 and were stained with calcofluor white. All cultures were grown at 37°C unless labeled otherwise. +, treatment with 0.5 U of chitinase for 3 h. FCZ, fluconazole (0.5 mg/liter); CFW, calcofluor white (50 mg/liter); CR, Congo red (2 mg/liter); SDS, sodium dodecyl sulfate (100 mg/liter). Scale bar, 10 μm.

Fig 5

Relative sedimentation times of different C. albicans mutant strains and wild-type cells cultured under various stress conditions. The sedimentation times of wild-type cells grown at 37°C without (Wt 37°C) or with 0.5 mg/liter fluconazole (FCZ) or 40 mg/liter calcofluor white (CFW), of wild-type cells grown at 42°C, or of various mutant strains at 37°C were compared. Strains that were treated with chitinase are indicated (+). The time needed for the optical density to decline to 80% of the initial optical density was determined and is expressed as a percentage of wild-type cells grown at 37°C (relative sedimentation time). The sedimentation time of wild-type cells was 16.2 min. Bars represent averages ± standard deviations of two experiments. *, P < 0.05; **, P < 0.01 (relative to the wild-type control at 37°C).