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. 2010 Apr;9(4):569-77.
doi: 10.1128/EC.00321-09. Epub 2010 Jan 29.

Farnesol induces hydrogen peroxide resistance in Candida albicans yeast by inhibiting the Ras-cyclic AMP signaling pathway

Aurélie Deveau  1 , Amy E Piispanen, Angelyca A Jackson, Deborah A Hogan
Affiliations

Farnesol induces hydrogen peroxide resistance in Candida albicans yeast by inhibiting the Ras-cyclic AMP signaling pathway

Aurélie Deveau et al. Eukaryot Cell. 2010 Apr.

Abstract

Farnesol, a Candida albicans cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, alpha-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate that farnesol induces oxidative-stress resistance by a similar mechanism. Strains lacking either Ras1 or Cyr1 no longer exhibited increased protection against hydrogen peroxide upon preincubation with farnesol. While we also observed the previously reported increase in the phosphorylation level of Hog1, a known regulator of oxidative-stress resistance, in the presence of farnesol, the hog1/hog1 mutant did not differ from wild-type strains in terms of farnesol-induced oxidative-stress resistance. Analysis of Hog1 levels and its phosphorylation states in different mutant backgrounds indicated that mutation of the components of the Ras1-adenylate cyclase pathway was sufficient to cause an increase of Hog1 phosphorylation even in the absence of farnesol or other exogenous sources of oxidative stress. This finding indicates the presence of unknown links between these signaling pathways. Our results suggest that farnesol effects on the Ras-adenylate cyclase cascade are responsible for many of the observed activities of this fungal signaling molecule.

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Figures

Fig. 1.
Fig. 1.
Role of CAT1 in farnesol-induced H2O2 survival. (A) Epifluorescence and differential interference contrast (DIC) microscopic views of CAT1-GFP cells in exponential phase treated with 50 μM farnesol for 2 h. (B) Histogram of fluorescence intensities of CAT1 in a farnesol-treated population of C. albicans cells with CAT1-GFP promoter fusion during early exponential-phase growth in liquid culture as revealed by laser scanning cytometry. Three subpopulations visualized on the histogram by the P4, P5, and P6 bars were sorted and challenged with 10 mM H2O2. LF, low fluorescence intensity (P6 subpopulation); MF, medium fluorescence (P5); HF, high fluorescence (P4). AU, arbitrary units. (C) Survival of the three subpopulations of cells shown in panel A under 10 mM H2O2 treatment. The data are expressed as the mean value (plus standard deviation [SD]) of duplicate samples. (D) Effect of pretreatment with 50 μM farnesol on the survival of cat1/cat1 and WT cells in H2O2. Following 2 h of incubation with 50 μM farnesol in YPD at 30°C, cells were harvested and challenged for 90 min with 0.5 mM (Δcat1/cat1) or 10 mM (WT) H2O2. Survival was assessed by dilution plating. The fold survival is expressed as the ratio between the survival of farnesol-treated cells and untreated cells. The data are expressed as the mean value (±SD) of three independent cultures. Survival after H2O2 exposure and pretreatment with farnesol was significantly higher in CU2 (WT), but not in the cat1/cat1 mutant (t test; P < 0.05).
Fig. 2.
Fig. 2.
Effects of farnesol, ascorbic acid, and α-tocopherol on ROS accumulation in cat1/cat1 cells as revealed by DCFH-DA staining. The cells were incubated in YPD at 30°C for 30 min with either 50 or 100 μM farnesol, 50 mM ascorbic acid, 50 μM α-tocopherol, or the appropriate vehicle control. The cells were then harvested, washed, and incubated with DCFH-DA for 30 min. Fluorescence was examined by epifluorescence microscopy with a fixed exposure time, and the quantification of cells accumulating ROS was performed by scoring the number of green fluorescent cells relative to all cells. The data are expressed as the mean value (plus SD) of triplicate samples.
Fig. 3.
Fig. 3.
(A) Effect of α-tocopherol on farnesol protection against H2O2. α-Tocopherol, farnesol, or both (50 μM) were added simultaneously to exponential-phase C. albicans SC5314 cultures, followed by incubation for 2 h. The cells were then challenged or not with 10 mM H2O2 for 90 min, and survival was measured by counting CFU. The data are expressed as the mean value (plus SD) of three independent cultures. (B) Quantification of CAT1 transcripts in C. albicans SC5314 culture. α-Tocopherol, farnesol, or both (50 μM) were added simultaneously to exponential-phase C. albicans SC5314 cultures, followed by incubation for 2 h. The transcript levels were normalized to GPD1 control transcript. The data are expressed as the mean value (plus SD) of three independent cultures.
Fig. 4.
Fig. 4.
Hog1 and Chk1 are not required for farnesol-mediated protection against H2O2 stress. Following 2 h of incubation with 50 μM farnesol in YPD at 30°C, cells were harvested and challenged for 90 min with 10 mM H2O2. Survival was assessed by dilution plating. The fold survival is expressed as the ratio between the survival of farnesol-treated cells and untreated cells. The parental hog1/hog1 and chk1/chk1 strains RM100 and CAF2 were used as WT controls. The data are expressed as the mean value (plus SD) of three independent cultures. (Inset) The phosphorylation of Hog1 in SC5314 cells was assessed by Western blotting after 30 min of incubation without any treatment (lane 1), with 10 mM H2O2 (lane 2), or with 50 μM farnesol (lane 3). The hog1/hog1 strain is shown as a control (lane 4).
Fig. 5.
Fig. 5.
(A) ras1/ras1 and cyr1/cyr1 mutants lack farnesol-mediated protection against H2O2 stress. Survival was assessed as described in the legend to Fig. 4. The fold survival is expressed as the ratio between the survival of farnesol-treated cells and untreated cells. The data are expressed as the mean value (plus SD) of three independent cultures. (B) Expression of CAT1 and GPD1 in C. albicans CAF2 and ras1/ras1 cultures in response to H2O2. Cells were challenged with 10 mM H2O2 for 30 or 60 min. The experiments were performed independently three times, and the RT-PCR is representative of the results obtained each time.
Fig. 6.
Fig. 6.
The Ras-cAMP cascade inhibits Hog1 phosphorylation. (A) Western blot of Hog1 and Hog1-P. Washed cells from overnight cultures of the wild-type CAF2, Δras1/ras1, Δcyr1/cyr1 Δras1/ras1/RAS1, and Δhog1/hog1 strains were resuspended in YPD and incubated for 2 h at 30°C. The CAF2 cells were then treated with 10 mM H2O2 (+) or water (−), and all strains were incubated for another 30 min. Cells were collected and processed for Western blot analyses of Hog1 and Hog1-P levels. Coomassie staining was used to visualize and normalize the total amounts of proteins. The experiments were performed independently at least three times, and the Western blot is representative of the results obtained each time. (B) Quantification of Hog1 protein (white bars) and Hog1 phosphorylation (black bars) levels in the different genetic backgrounds. The levels are expressed relative to the intensity measured in the control treatment. All data were normalized to Coomassie-stained SDS-PAGE protein levels. The data are expressed as the mean value (plus SD) of two independent experiments.
Fig. 7.
Fig. 7.
(A) Proposed model for the mechanism of farnesol protection against OX stress. (B) Interaction between farnesol-associated signaling pathways (2, 12, 33–35, 49, 50).
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