Hydrogen peroxide induces hyphal differentiation in Candida albicans

Abstract

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H(2)O(2) in Candida albicans. This finding is confirmed by the changing intracellular concentration of H(2)O(2). In order to induce the same level of differentiation, low concentrations of exogenous H(2)O(2) are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.

FIG. 1.

Hyphal induction by exogenous H₂O₂. (A) Microscopic images of H₂O₂-induced hyphae. Wt cells were grown on YPD solid plates supplemented with the indicated concentrations of H₂O₂ at 30°C for 6 days. Representative colonies were photographed with a stereomicroscope (top). Cells in the mid-log phase were cultured in YPD liquid medium containing H₂O₂ for 6 h at 30°C and observed with a light microscope (bottom). (B) Cytotoxicity of H₂O₂. Standardized cell suspensions were challenged with the indicated concentrations of H₂O₂ for 30 min, plated onto YPD solid medium, and incubated at 30°C for 2 days. The survival rate was expressed as a percentage of the number of colonies in the presence of H₂O₂ divided by the number of colonies in the absence of H₂O₂. (C) Efficiency of hyphal differentiation. Cells were grown on YPD solid medium containing the indicated concentrations of H₂O₂ and incubated at 30°C for 6 days. The percentage of hyphal differentiation was expressed as the number of hyphal colonies divided by the total number of colonies.

FIG. 2.

Construction of CAT1 null mutants and a revertant. The CAT1 genes of the wt and the tsa1Δ mutant (30, 31) were disrupted using URA3-dpl200 (32), yielding the cat1Δ and tsa1Δ cat1Δ mutants, respectively. The sense and antisense primers were nucleotide positions 754 to 823 and 2312 to 2381, respectively, of the CAT1 open reading frame (ORF). To construct a revertant, the DNA fragment containing its own promoter, ORF, and terminator was cloned into pLUX, linearized with NheI, and transformed into the cat1Δ mutant. Southern (A) and Northern (B) analyses were performed to confirm the authenticity of the constructed strains, using the ³²P-labeled probe prepared from the MfeI fragment of the CAT1 ORF. For the Southern analyses, genomic DNA was digested with NsiI and NcoI. Lanes 1, parental strains (CAI4 and the tsa1Δ mutant in panels A and B, respectively); lanes 2, strains with one allele disrupted; lanes 3, strains with URA3 popped out from the lane 2 strains; lanes 4, null mutants (the cat1Δ and tsa1Δ cat1Δ mutants in panels A and B, respectively); lanes 5, strains with URA3 popped out from the cat1Δ mutant; lanes 6, CAT1-reintroduced strains of the cat1Δ mutant.

FIG. 3.

Effects of increased intrinsic H₂O₂ on hyphal differentiation. Cells were grown in YPD medium containing 0.2 and 1 mM H₂O₂ for 6 h, washed, and resuspended in Hank's balanced salt solution. After the addition of CM-H2DCFDA (10 μM final), the cells were further incubated at RT for 10 min. (A) Images of DCF fluorescence were taken by using a confocal microscope with excitation and emission wavelengths at 488 nm and 520 nm, respectively. (B) Relative concentrations of intracellular H₂O₂ were derived from the confocal microscope-aided integration of fluorescence signal intensity within a scope. (C) Efficiency of hyphal differentiation at 0.2 mM H₂O₂ was determined as described in the legend to Fig. 1C. CAT1-R represents the strain into which the functional CAT1 gene was introduced.

FIG. 4.

Effects of ascorbic acid on hyphal differentiation in FBS-treated cells. Wt cells were grown in YPD in the absence (−) or presence (+) of 10% FBS for 30 min, followed by supplementation with 50 mM or 100 mM ascorbic acid. A portion of the cells was removed to take light microscopic images (A). For the rest of cells, fluorescence images (B) were taken, and the relative concentrations of intracellular H₂O₂ (C) were determined as described in the legend to Fig. 3.

FIG. 5.

Effects of ascorbic acid on hyphal differentiation. After the wt and tsa1Δ, cat1Δ, and tsa1Δ cat1Δ mutant cells were grown in YPD medium supplemented with 4 concentrations of exogenous H₂O₂ for 30 min at 30°C, 100 mM ascorbic acid was added, and the cells were further grown for 6 h at 30°C, the cultures were observed with a light microscope (magnification, ×400).