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Edited by Michael H. Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved November 30, 2005 (received for review July 27, 2005)
Abstract
A better understanding of the molecular basis of programmed cell death (PCD) in fungi could provide information that is useful in the design of antifungal drugs that combat life-threatening fungal infections. Harsh environmental stresses, such as acetic acid or hydrogen peroxide, have been shown to induce PCD in the pathogenic fungus Candida albicans. In this study, we show that dying cells progress from an apoptotic state to a secondary necrotic state and that the rate at which this change occurs is proportional to the intensity of the stimulus. Also, we found that the temporal response is modulated by Ras–cAMP–PKA signals. Mutations that block Ras–cAMP–PKA signaling (ras1Δ, cdc35Δ, tpk1Δ, and tpk2Δ) suppress or delay the apoptotic response, whereas mutations that stimulate signaling (RAS1val13 and pde2Δ) accelerate the rate of entry of cells into apoptosis. Pharmacological stimulation or inhibition of Ras signaling reinforces these findings. Transient increases in endogenous cAMP occur under conditions that stimulate apoptosis but not growth arrest. Death-specific changes in the abundance of different isoforms of the PKA regulatory subunit, Bcy1p, are also observed. Activation of Ras signals may regulate PCD of C. albicans, either by inhibiting antiapoptotic functions (such as stress responses) or by activating proapoptotic functions.
The fungal pathogen Candida albicans causes a range of clinical conditions, including superficial but nonetheless irritating infections of the oral and vaginal mucosa (thrush) to life-threatening systemic disease in immunocompromised patients (1). Over a lifetime, 80% of women suffer a Candida infection, and in ≈5% of these cases, thrush is a recurrent problem (2). Systemic candidiasis occurs in patients undergoing chemotherapy or organ transplantation and, depending on the patient group, one-third to one-half of these infections can be fatal. The levels of infection among premature low-birth-weight babies can be as high as 7%, and more than one-half of these infections are fatal (3). The number of clinical C. albicans infections worldwide has risen considerably in recent years (4, 5), and the incidence of resistance to traditional antifungal therapies is also rising (6). Many existing antifungal therapies have unfortunate clinical side effects, including severe nephrotoxicity; therefore, strategies are needed to identify targets for antifungal therapy.
Cell death accompanied by the hallmark features of apoptosis has been observed in Saccharomyces cerevisiae after exposure to a range of different environmental stresses, including, for example, low fungicidal doses of hydrogen peroxide or acetic acid (7, 8). In S. cerevisiae, dying yeast cells may undergo distinct physiological changes, including chromatin condensation along the margin of the nuclear envelope, nuclear fragmentation, delayed loss of plasma membrane potential, and the exposure of phosphatidylserine on the outer surface of the plasma membrane. Also, yeasts appear to contain the key ancestral components that are necessary to initiate and complete a programmed cell death (PCD) response akin to apoptosis. Several functionally related components of the mammalian apoptotic cascade, including an apoptosis-inducing factor (AIF1) and a yeast metacaspase (MCA1) (reviewed in refs. 9 and 10), have been described in yeasts.
PCD in unicellular organisms such as yeasts might appear initially to result in the death of the whole organism and, therefore, have no adaptive value. However, single-celled organisms are rarely found as single entities. Instead, they exist alongside their clonal relatives where altruistic cell suicide might have favorable evolutionary repercussions (e.g., by limiting the spread of selfish genetic elements or regulating population density) (11–13).
We have demonstrated (14) that C. albicans can be triggered to undergo an apoptotic cell-death response when exposed to oxidative stress, intracellular acidification, or low fungicidal doses of amphotericin B. An understanding of the mechanistic basis of cell-death decisions in this organism could lead to the development of antifungal agents that work by activating endogenous fungal cell suicide mechanisms. With this aim in mind, we set out to identify the molecular components of the PCD response in C. albicans.
In this study, we investigated the role of the Ras–cAMP–PKA signaling pathway in the PCD response of C. albicans. In mammals, Ras exhibits both proapoptotic and antiapoptotic functions (15–18), but nothing is known of its role in the death response of C. albicans. Ras signaling during morphogenesis of C. albicans has been studied extensively (19, 20), and, as with Ras in S. cerevisiae, it regulates both mitogen-activated protein kinase-dependent and cAMP–PKA-dependent responses. The MAPK responses depend on the transcription factor CPH1 (20), and the cAMP–PKA responses depend on the transcriptional regulator EFG1 (20–23). From this study, we conclude that Ras signals in C. albicans also have a role in the acceleration of PCD events, which involves the production of cAMP signals; however, because Ras–cAMP–PKA activation per se is not sufficient to bring about cell death, we cannot exclude the possibility that Ras may also exert its effects through other pathways.
Results
Temporal Analysis of Death Response: Apoptotic Yeast Cells Become Necrotic. Our previous studies revealed that C. albicans (CAF2-1) undergoes an apoptotic cell-death response when treated with low fungicidal doses of acetic acid or hydrogen peroxide (14), whereas higher doses produced a greater percentage of necrotic cells. These findings were based on the examination of cells after exposure to the death-inducing agents for just 200 min. To gain a greater understanding of the temporal progression of cell death in C. albicans, CAF2-1 cells were exposed to treatments we had previously determined to be proapoptotic or pronecrotic. The cells were then examined at a range of time points for overall viability, double-stranded DNA breaks (by using the TUNEL assay), and the ability to exclude propidium iodide (PI) (Fig. 1).
Fig. 1.
Necrosis follows apoptosis in C. albicans. The numbers of live, apoptotic, and necrotic cells were assessed at different time points in WT C. albicans strain CAF2-1. (A) Apoptosis (TUNEL-FITC) and necrosis (PI uptake) shown in cells that were treated with 300 mM acetic acid in SC (pH 3.0) at different times. BF, bright field; DIC, differential interference contrast. (B) Change in percentage of TUNEL (+) cells over time after treatment with different doses of acetic acid (black, 0 mM control treatment; green, 20 mM nonlethal stress; blue, 120 mM low-dose fungicidal; and red, 300 mM high-dose fungicidal). Temporal responses of WT death in 120 mM acetic acid (C), 300 mM acetic acid (D), and 25 mM hydrogen peroxide (E).
Cells that were grown in synthetic complete (SC) medium (pH 3.0) and treated with low fungicidal dose of acetic acid or hydrogen peroxide (120 mM acetic acid or 5 mM H2O2) for 210 min were predominantly apoptotic as revealed by both TUNEL staining and PI exclusion (Fig. 1 B and C ). At higher doses, e.g., 300 mM acetic acid or 25 mM H2O2, most cells were necrotic after 210 min of treatment (Fig. 1 B, D, and E ). However, cells examined at earlier time points were mostly apoptotic. For example, when cells were examined after incubation for 120 min in the presence of 300 mM acetic acid, 85% of cells appeared to be apoptotic according to the PI exclusion assay and 90% apoptotic by the TUNEL assay (Fig. 1 A ). However, by the 210-min time point, 82% of the cells were necrotic by the PI exclusion assay, and the numbers of TUNEL-positive cells fell to 55%. A similar trend was observed after the treatment of cells with 25 mM hydrogen peroxide; 46% of cells were apoptotic at the 45-min time point, but 85% were necrotic by 210 min. Very few PI or TUNEL staining cells were observed at any time when cells were treated with stress-inducing doses of acetic acid (20 mM) or hydrogen peroxide (1 mM), even though growth had been completely suppressed. Primary (immediate) necrosis was observed in only a small percentage of acetic acid-treated cells (typically, 2–3%) up to a maximum of 20% in cells treated with the highest doses of hydrogen peroxide (25–50 mM). At critical doses of the tested death-inducing agents, cells appeared to initiate a PCD response, die in an apoptotic manner, and then proceed to become necrotic as a secondary consequence.
ras1Δ and cdc35Δ Mutant Cells Are More Resistant to Death-Inducing Stimuli than WT Cells. The Ras–cAMP–PKA pathway has been studied extensively in C. albicans. The Ras1 protein activates adenylate cyclase, Cdc35p (21, 23), which generates cAMP in response to morphogenetic stimuli. The regulatory subunit of PKA is encoded by BCY1 (24) and the catalytic subunit by two functionally redundant genes TPK1 and TPK2 (22, 25). The downstream targets remain largely unknown, although they may include Efg1 (26, 27).
We examined the viability of WT, ras1Δ and cdc35Δ cells after treatment with 120 mM acetic acid or 50 mM hydrogen peroxide (Fig. 2 A and B ). As expected, essentially all WT cells were dead after exposure to either of these treatments after 200 min. However, 49% of ras1Δ cells survived the acetic acid treatment and 22% of mutant cells survived the hydrogen peroxide treatment. These data suggest that ras1Δ cells are more resistant to lethal stimuli than WT cells. Analysis of cdc35Δ cells after exposure to these two treatments revealed that this mutation confers an even greater resistance to cell death: 66% of cdc35Δ cells survived the acetic acid treatment and 81% survived the hydrogen peroxide treatment. To determine whether Ras1p or Cdc35p-deficient cells can undergo apoptosis we exposed ras1Δ and cdc35Δ cells to a higher dose of 240 mM acetic acid for 200 min. 99.9% of ras1Δ cells and 99.4% of cdc35Δ cells were killed by this treatment; however, 43.3% and 56.7% of the cells, respectively, had retained the ability to exclude PI, indicative of apoptosis (data not shown). This finding implies that both ras1Δ and cdc35Δ cells can still be induced to undergo apoptosis, albeit at higher concentrations of death-inducing agent.
Fig. 2.
Genetic inactivation of Ras pathway abrogates killing, whereas activation of the Ras pathway accelerates killing. Survival of ras1Δ, cdc35Δ, and WT control after exposure to 120 mM acetic acid (A) or 50 mM hydrogen peroxide (B) for 200 min. Cell viability was based on CFUs formed from plating 1,000 cells in triplicate of each treated population at the lowest dilution. Data represent the mean ± SE of three independent experiments. Percentage of cells that are apoptotic (black bars) and necrotic (white bars) after exposure to 120 mM acetic acid for different times is shown for WT control cells (C) and RAS val13 cells (D).
These results implicate the Ras/cAMP signaling module as being important in active cell death. The downstream target of the Ras/cAMP signaling cascade is PKA, the catalytic subunits of which are encoded by TPK1 and TPK2 (25). It is predicted that disruption of PKA would also result in resistance to death-inducing conditions. However, PKA activity appears to be essential for viability, and thus, it has not been possible to test the phenotype of a double mutant. Analysis of single TPK mutants showed no significant difference from WT cells (data not shown) which suggests that there is sufficient redundancy between the subunits to mask the role of individual Tpk1p or Tpk2p defects with respect to a function in apoptosis. It also cannot be ruled out at this stage that Ras may also be exerting death dependent effects through other pathways.
Hyperactivation of the Ras Pathway Accelerates Cell Death. The Ras-signaling pathway in C. albicans can be hyperactivated by dominant RAS1 mutations, such as RAS1val13 (19). We integrated a Ras1val13 construct (and the vector control) in the same genetic background as the other mutants we have used in this study and tested their ability to undergo apoptosis-like death. Hyperactivation of the RAS1val13 allele, under the control of the MAL2 promoter (in the presence of sucrose) resulted in the appearance of wrinkly colonies on solid medium as expected (19) (data not shown). To assess the effect of Ras hyperactivation on the cell-death response, both the control and RAS1val13 strains were grown in yeast–peptone (YP) broth containing 2% glucose (YPD) and resuspended in YP–sucrose at OD600 of 0.125 and incubated for approximately two doubling times. Cells were then resuspended in YP–sucrose at pH 3.0, and the numbers of live, apoptotic, and necrotic cells assessed at different times after acetic acid treatment. Doubling times of the control and RASval13 strains during these experiments were identical on sucrose or glucose (1.1 h).
After 50 min of treatment with 120 mM acetic acid, 72% of the WT cells were viable compared with only 19% of the RAS1val13 cells (Fig. 2 C and D ). This result is consistent with the notion that elevated signaling through the Ras pathway accelerates cell death, by promoting apoptosis. As seen previously in WT, the RAS1val13 apoptotic cells eventually became necrotic. However, the overall time for cells to progress from an apoptotic to a necrotic state was not significantly affected by hyperactivation of Ras1, suggesting that this progression is independent of Ras activity.
Pharmacological Manipulation of Ras–cAMP Signals Modulate Cell-Death Responses. In addition to hyperactivating the Ras pathway using genetic approaches, the effects of pharmacological manipulation of the pathway were also assessed. WT cells were grown and treated with 0–120 mM acetic acid, in the presence or absence of dibutyryl cAMP (db.cAMP), caffeine, forskolin, dideoxyforskolin, or lovastatin, and the appropriate drug vehicle controls, water or 0.2% DMSO. No significant differences could be detected between cells treated with acetic acid in the presence of water alone, DMSO or 7 μM dideoxyforskolin. Cells treated with db.cAMP were more sensitive to acetic acid, with only 29% of cells being viable after incubation for 50 min, compared with 70% viability in the absence of db.cAMP (Fig. 3 A and B ). This result is consistent with the data from the analysis of the RAS1val13 mutant cells, with activation of the pathway accelerating entry into PCD. Two other cAMP-stimulatory drugs (5 mM caffeine targeting phosphodiesterase and 7 μM forskolin activating adenylate cyclase) also promoted apoptotic cell death in response to acetic acid. This finding was supported further by the observation that both acetic acid, and hydrogen peroxide induced killing were subtly enhanced (data not shown) in a phosphodiesterase mutant strain (pde2Δ) that contains elevated levels of cAMP (28). Treatment of cells with acetic acid and 25 mM lovastatin (which blocks Ras farnesylation and its membrane localization), decreased the amount of apoptotic cell death observed (Fig. 3C ). The drugs did not promote apoptosis in the absence of acetic acid, indicating that Ras–cAMP–PKA pathway activation per se is not sufficient to bring about PCD.
Fig. 3.
Drugs that affect Ras–cAMP–PKA signaling influence the mode of killing. Effects of db.cAMP on proportion of WT cells that are apoptotic (black bars) and necrotic (white bars) after exposure to 120 mM acetic acid for different times. (A) Without db.cAMP. (B) With db.cAMP. (C) Effects of db.cAMP, caffeine (Caf), forskolin (F), dideoxyforskolin (ddF), and lovastatin (L) on the overall level of apoptotic cell death in WT cells treated with 80 mM acetic acid for 200 min. Water or DMSO were used as vehicle controls for drug treatments. ***, P < 0.001, compared with control (two-tailed t test; n = 300 cells for each treatment). nsd, not significantly different.
cAMP Signals Are Activated During PCD. The levels of endogenous cAMP in WT cells were assessed at various time points after the induction of apoptosis. Fig. 4A shows that the level of cAMP increased rapidly for ≈10 min after the treatment. After the initial rise the levels fell back to slightly below baseline levels within 20 min of treatment. Over the subsequent hours, a more chronic rise in cAMP level was observed. Similar changes in cAMP were observed when cells were treated with 40 μM phytosphingosine (data not shown), another drug that has been shown to induce apoptosis in yeasts and filamentous fungi (29). The levels of cAMP in control cells remained relatively constant throughout the experiment. Also, levels of cAMP in cells treated with nonlethal, but stress-inducing doses of acetic acid behaved just like the untreated controls, indicating that the increase is related to the death response and not simply growth inhibition.
Fig. 4.
Changes in cAMP and BCY1p accompany PCD in C. albicans.(A) cAMP in WT CAF2-1 cells grown at 30°C in YPD (pH 3.0) and then exposed to 0 mM acetic acid (open squares indicate control) or a lethal dose of 40 mM acetic acid (filled squares indicate 95% apoptotic cells at 200 min of assay). Reduced levels of Bcy1p isoforms were observed after 1 h of acetic acid exposure during PCD but not stress responses of WT C. albicans.(B) Bcy1p isoforms (1 and 2) on 2D gels under the following different treatment conditions: 0 mM control, 20 mM stress, 120 mM apoptosis, and 300 mM necrosis-inducing after 200 min of treatment. (C) Normalized volumes of Bcy1p isoforms after treatment. Normalized volumes are shown based on four samples ± SE. Significance difference compared with control was determined by two-tailed heteroscedastic t tests (df = 6). Bcy1 (1) and Bcy1 (2) at 20 mM acetic acid were not significantly different; Bcy1 (1) at 120 mM, P = 0.000863; Bcy1 (2) at 120 mM, P = 0.0294; Bcy1 (1) at 300 mM, P = 0.0657; and Bcy1 (2), P = 0.0172.
Bcy1p Levels Fall During PCD. To further understand the effects of the Ras–cAMP–PKA pathway on modulating the rate of cell death in C. albicans we wanted to investigate the effects of up-regulating the Ras–cAMP–PKA signal transduction module by knocking out BCY1, the regulatory subunit of PKA. In S. cerevisiae deletion of BCY1 is nonlethal (30), and it leads to constitutive cAMP-independent activation of PKA. However, it appears that deletion of BCY1 in C. albicans is lethal (24). This finding is consistent with our prediction that up-regulating the Ras–cAMP–PKA pathway in C. albicans will increase entry into cell death. Because no viable knockout of BCY1 can be made in a CAI4 background of C. albicans, we decided to look at the levels of this protein by using 2D gel electrophoresis.
Several different isoforms of Bcy1p have been reported in S. cerevisiae (31, 32). We have identified two isoforms of C. albicans Bcy1p on 2D gels. These isoforms have similar molecular masses of ≈57,500 Da [Bcy1p (1)] and 57,800 Da [Bcy1p (2)], which are slightly higher than the mass predicted on sequence data alone of 50,306 Da, and they have distinct pIs of ≈5.12 and 5.24, respectively (Fig. 4B ).
To examine the response of the Bcy1 protein to stress and death-inducing stimuli, proteins isolated from cells treated with 0, 20, 120, and 300 mM acetic acid were separated by 2D gel electrophoresis and the abundance of the Bcy1 isoforms quantified after 1 h treatment (Fig. 4B ). The normalized abundance (Fig. 4C ) of both Bcy1p (1) and Bcy1p (2) remained constant in growing cells (0 mM acetic acid) and growth arrested but viable cells (20 mM acetic acid), but both isoforms declined significantly in abundance when treated with higher fungicidal doses (120 or 300 mM acetic acid).
Discussion
Ras signals play an important role in the determination of cell fate (proliferation, differentiation and death) in a number of animal models. However, the situation is quite complex because activation of Ras can have either proapoptotic or antiapoptotic functions according to the cell type (15–18). This complexity is mirrored to a certain extent in fungi as well.
We have shown that the Ras–cAMP signaling pathway plays an important role in the PCD response of C. albicans induced by acetic acid and hydrogen peroxide. Artificial Ras activation accelerates the rate at which cells become apoptotic, but Ras activation in itself is not sufficient to kill cells. Disruption of Ras signaling inhibits the rate of entry into apoptosis, but does not prevent it from happening if the stimulus is sufficiently strong. Necrosis ensues as a secondary consequence of apoptotic cell death and appears to be independent of Ras signaling.
Ras appears to have a predominantly antiapoptotic role in hyphae (but not yeasts) of the Zygomycete fungus Mucor racemosus, because lovastatin, an indirect inhibitor of Ras prenylation, induces apoptotic cell death (33). Exogenous application of dibutyryl cAMP (which can initiate morphogenesis from hyphal to yeast-like growth) partially reduces the lovastatin-induced cell-death response. Lovastatin alone, at the doses we tested, had no effect on cell fate in C. albicans.
In S. cerevisiae, reactive oxygen species production, the expression of antioxidant proteins and the apoptotic response that follows treatment with osmotin are partially dependent upon an induced suppression of stress responses via the RAS2-signaling pathway (34). RAS2 G19V, a dominant active allele of RAS2, increases sensitivity to osmotin and a null mutant decreases sensitivity. The response is linked specifically to Ras–PKA, rather than Ras–mitogen-activated protein kinase signaling, because the effects of the dominant active allele are also seen in a ste20 background. Consistent with osmotin induced Ras–PKA signaling, a bcy1 null, with a constitutively active PKA activity, significantly increases sensitivity to osmotin.
Several differences are apparent between the Ras–cAMP–PKA pathways in S. cerevisiae and C. albicans. Most notable in this respect is the finding that deletion of the sole Ras encoding gene, RAS1 in C. albicans is viable, whereas the double ras1 ras2 null in S. cerevisiae is lethal (35). Conversely, BCY1 disruption in S. cerevisiae is viable (36), whereas the constitutive activation of PKA by BCY1 deletion in C. albicans has not been possible (24). There are clearly differences between these fungi in the way these pathways operate, which may be attributable to differences in their targets, cross-talk between signaling pathways and functional reassignments.
For example, derepression of stress-response element (STRE)-dependent transcriptional responses in ras2 mutants (37) could account for the elevated resistance of RAS2 nulls to stress treatments. During osmotin induced PCD, both STRE element and Yap1p-response element (YRE) reporter constructs are repressed (34), and thus, it might be argued that the balance between stress and apoptotic signals ultimately determines cell fate. Although osmotin-induced PCD in WT cells is associated with a suppression of STRE signaling, this situation cannot be generalized to other PCD responses, because at similar levels of apoptotic cell death, hydrogen peroxide still stimulates STRE reporter expression (34). However, it may be concluded that osmotin stimulates proapoptotic reactive oxygen species production by means of the activation of the Ras2-signaling pathway, which inhibits YRE (Yap1-dependent) and STRE-mediated antioxidant stress responses. Gourlay and Ayscough (38) found that PDE2 overexpression in S. cerevisiae rescued end3Δ cells (with defects in actin organization that typically lead to a hypersensitive H2O2-induced apoptotic response) in a PKA-independent manner, which is evidence that cAMP effects on cell death can be more direct, by-passing the PKA-mediated inhibition of the general stress response. Although our data indicate that death responses are affected by altering Ras–cAMP signals, we do not exclude the possibility that these effects may be PKA-independent under some circumstances. This possibility is an important consideration because, in C. albicans, no role has yet been found for STRE in the regulation of stress responses (39, 40), which may have been reassigned for other roles, such as morphogenesis (41).
A common theme in fungal responses to stress is an interrelationship between cell death and morphogenesis or differentiation. As shown by Narasimhan et al. (34), overexpression of plant-defense molecules induces hyperbranching or formation of spiral hyphae in fungi (42), and growth inhibition per se is associated with altered hyphal branching (43, 44). The Ras pathway has been strongly linked to morphogenetic signals in a number of fungi (45). As we have shown, RAS1 deletion in C. albicans is able to decelerate, or even prevent, PCD just as it is in an S. cerevisiae ras2 strain, and it has an important role in activating the yeast-to-hypha transition (19). Whereas ste12 knock-outs in S. cerevisiae are defective in their apoptotic response (34), disruption of known downstream effectors of the Ras pathway in C. albicans, CPH1 or EFG1, appears to have no effect (14). Although Ras activation could lead to the induction of hyphae, we have observed in dying C. albicans that it must operate through other unidentified pathways, because the switching is also independent of RIM101 and TEC1 (14).
The downstream effectors of the Ras-mediated cell-death response in C. albicans have yet to be identified. Drugs that target fungal Ras–cAMP–PKA signals might be used to enhance the efficacy of traditional antifungal therapies by blocking antiapoptotic stress responses or activating proapoptotic functions, perhaps turning fungistatic drugs into more attractive fungicidal drugs. Promoting the onset of fungal apoptosis could improve the outlook for patients with recurrent infections or life-threatening systemic disease.
Materials and Methods
Strains, Plasmids, Media, and Treatments. All C. albicans strains were derivatives of SC5314. CAF2-1 our standard WT was constructed by Fonzi and Irwin (46) as a Ura+ control of the genetically tractable CAI4 URA– strain. The genotypes of the mutants used in this study were as follows: WT, CAF2-1 (Ura+) and CAI4 (Ura–) (46); Ras1Δ, ras1.::hisG/ras1.::hphURA3-hphura3.:λimm434/ura3.::λimm-434 (15); Cdc35Δ, cdc35.:: hisG/cdc35.::hisG-UR A3-hisG ura3.::λimm434/ura3.::λimm-434 (23); Tpk1Δ, tpk1.::hisG/tpk1.::hisG-URA3-hisGura3.::λimm434/ura3.::λimm434; and Tpk2Δ, tpk2.::hisG/tpk2.::hisG-URA3-hisGura3.::λimm434/ura3.::λimm434 (22). The regulatable-RAS1 val13 strain and its control RAS1 val13 -VECT alone strain were made in CAI4 during this study. The hyperactivated RAS1val13 allele and a control vector only plasmid were obtained from the Fink laboratory (15). The two plasmids were cut with AscI to produce linear fragments and then transformed independently into C. albicans (CAI4). Correct integration of the plasmids was confirmed by Southern blot analysis.
All C. albicans strains were grown in YPD or SC, as required. For treatment of cells under weak acid stress or death promoting conditions, media were prepared at pH 3.0 and then supplemented with acetic acid at appropriate doses. For treatment with hydrogen peroxide, cells were grown in YPD and then transferred to media containing hydrogen peroxide at the required concentration. To induce expression of RAS1val13 , cells were grown in YPD and then switched to YP plus 2% sucrose or 2% maltose before treatment.
All treatments were applied to cells at a density of 1 × 107 cells per ml. Most of the data presented are for cells growing in YPD, but for the proteomics analysis experiments were performed in SC (pH 3.0). Higher doses of acetic acid were required to bring about the same amount of killing when cells were grown in SC (pH 3.0) rather than YPD (pH 3.0).
Pharmacological Manipulation of Ras–cAMP signaling. CAF2-1 cells were grown to a density of 1 × 107 cells per ml in YPD (pH 3.0) and treated with water or DMSO (final concentration of <0.2%) as vehicle controls or with db.cAMP (in water), caffeine (in water), forskolin (in DMSO), dideoxyforskolin (in DMSO), or lovastatin (in DMSO). After incubation for 15 min at 30°C, cells were further treated with acetic acid to give final doses of 0, 40, 80, and 120 mM. Cell viability and PI staining were examined after 200 min of incubation at 30°C. Each experiment was performed in triplicate.
Death Assays. Overall viability was assessed by using clonogenic assays; cells at 1 × 107 cells per ml were diluted in series and plated in triplicate on YPD and then incubated at 30°C for 48 h. Necrosis was assessed by looking at PI uptake of cells using PI at 20 μg·ml–1. The proportions of apoptotic cells in treated populations were ascertained according to the single-cell method as described in ref. 14 and by TUNEL assays. All experiments were performed in triplicate on independent occasions.
cAMP Determination. C. albicans (CAF2-1) cultures were inoculated at 5 × 105 cells per ml in YPD (pH 3.0) and allowed to grow to a density of 2 × 107 cells per ml. Cells were then resuspended in YPD (pH3.0) before the addition of either acetic acid (final concentration 40 mM) or a water placebo. Cells were incubated at 30°C with shaking (200 rpm, Infors Multitron, Bottmingen, Switzerland) and 5-ml subsamples were removed at timed intervals over a 20-h period. Cells were captured on Whatman glass-fiber filters (GF/C, 25 mm in diameter) by rapid filtration and then immediately immersed in liquid nitrogen. After all samples had been collected and stored at –80°C, the cells were thawed in 1 ml of 1 M perchloric acid and freeze-fractured by repeated transfer between liquid N2 and a 65°C water bath. Samples were then processed according to a method described by Thevelein et al. (47). cAMP levels were determined by using the [3H]cAMP determination kit (TRK 432; Amersham Pharmacia, Little Chalfont, UK). Each assay was performed in duplicate according to the manufacturer's instructions.
2D Gel Electrophoresis. C. albicans (CAF2-1) cells were suspended in 100 ml of SC (pH 3.0) at a density of 2 × 107 cells per ml and treated as follows. Control cultures were treated with water, and the remaining aliquots were treated to give final doses of 20, 120, and 300 mM acetic acid. Cells were incubated at 30°C for 1 h and then frozen in liquid N2. Cell viability was determined at 200 min.
Proteins were extracted by bead beating the samples on ice in 160 μl of lysis solution containing 7.5 M urea, 2.5 M thiourea, 1.25 mM EDTA, 1.75 μg·ml–1 pepstatin A, and protease inhibitor mixture (Roche Diagnostics, Lewes, UK). After shearing, 40 μl of 20% (wt/vol) CHAPS, 50% (vol/vol) glycerol, and 10% (vol/vol) carrier ampholyte were added; the ice-cold samples were then vortexed for 6 × 30 s; and the supernatants were collected after a spin at 10,000 × g for 10 min at 4°C. Extracts were stored at –80°C until required. Protein samples (500 μg) were separated on IPGphor strips (pH 4–7) by using a step protocol (200 V for 1 h, 500 V for 1 h, and 1,000 V for 1 h) and a gradient switch of 1,000–8,000 V for 30 min and 8,000 V for 12 h (total of 80,000 V/h). For the second dimension, the strip gels were run on precast, 26 × 20-cm 12% SDS/PAGE in an Ettan Dalt II (Amersham Biosciences) electrophoresis tank at 25°C for 1 h at 2.5 W per gel and then separated at 19 W per gel until the dye front reached the gel bottom. All gels were run in quadruplicate. Gels were stained with Coomassie blue and then scanned at a resolution of 300 dots per inch (16-bit). Gels were dried and stored until required for spot identification by mass spectroscopy. Protein spots were cut from gels and digested with trypsin. Peptide masses were determined by using a PerSeptive Biosystems Voyager-DE STR mass spectrometer (MALDI-TOF).
Comparative spot-pattern analysis was performed by using phoretix 2D advanced software (Nonlinear USA, Durham, NC). Spot identifications were made by comparison with reference C. albicans 2D gels annotated by cogeme (Cogeme Proteome Service Facility 1, Aberdeen, Scotland). mascot software (Matrix Science, Boston) was used to compare the MALDI-TOF fingerprints of spots with hypothetical tryptic digests of the C. albicans genome sequence.
Acknowledgments
We thank G. R. Fink (Whitehead Institute, Cambridge, MA), L. Stateva (UMIST, Manchester, UK), J. Ernst (Heinrich-Heine University, Düsseldorf, Germany), and M. Whiteway (Biotechnology Research Institute, National Research Council, Montreal) for kindly providing us with strains; Al Brown for his continued support of our work into C. albicans and critical reading of the manuscript; and COGEME for assistance with the proteome analysis. M.R. was supported by a fellowship from the Lloyd's Tercentenary Foundation and by the University of Aberdeen. A.J.P. was supported by a Medical Research Council studentship. J.C. was funded by a Scottish Hospitals Endowments Research Trust grant (to M.R.).
Footnotes
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↵ * To whom correspondence should be addressed. E-mail: m.ramsdale{at}abdn.ac.uk.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: PCD, programmed cell death; PI, propidium iodide; SC, synthetic complete; db.cAMP, dibutyryl cAMP; YP, yeast–peptone; YPD, YP broth containing 2% glucose.
- Copyright © 2006, The National Academy of Sciences
References
Ras pathway signaling accelerates programmed cell death in the pathogenic fungus Candida albicans
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