Cyclic AMP and Fluconazole Resistance in Saccharomyces cerevisiae

Abstract

Cyclic AMP (cAMP) is involved in the stress response in Saccharomyces cerevisiae. We show that cAMP is required for resistance to fluconazole in S. cerevisiae. In addition, activation of Ras2, a regulator of cAMP generation, results in some protection from fluconazole toxicity in a fashion independent of the efflux transporter Pdr5p.

Saccharomyces cerevisiae, a genetically tractable fungus related to Candida albicans, is an attractive organism for the study of fluconazole resistance (4). Despite advances in understanding the mechanisms of fluconazole resistance in S. cerevisiae, however, the components of the response pathways are not fully known.

In S. cerevisiae, the RAS1 and RAS2 genes are activators of adenylate cyclase, which catalyzes the formation of cyclic AMP (cAMP), a central signaling molecule (10). Ras1p and Ras2p are highly homologous to the mammalian ras proteins (10). Activation of mammalian ras has been shown to affect the sensitivity of tumors to various antineoplastic agents via activation of the MDR-1 (multidrug resistance) gene, which is the mammalian homologue of PDR5 (1, 3). Since an important mechanism of fluconazole resistance in S. cerevisiae is mediated by PDR5 (pleiotropic drug resistance 5), an efflux transporter (2, 4), we examined whether cAMP and activation of the Ras pathway in S. cerevisiae similarly affect the resistance to fluconazole.

cAMP is required for resistance to fluconazole.

Standard procedures to prepare the media and to manipulate S. cerevisiae were used (5). To test whether cAMP has a function in resistance to fluconazole we used a strain in which internal cAMP levels can be changed by the addition of cAMP to the medium (strain SR 959 [MATa/α ras1/ras1 ras2/ras2 pde2/pde2] [7]). Thus, we can mimic functions of Ras connected to changing cAMP levels in the cell by changing external cAMP concentrations (7). Despite its very low intracellular cAMP level, the SR959 mutant does not require external cAMP for growth on synthetic complete (SC) medium (7). We compared the sensitivity of strain SR959 to fluconazole with that of the isogenic wild type (SR607 [7]) in the presence or absence of cAMP. As shown in Table 1, SR959 was very sensitive to fluconazole (100 μg in a paper disk by applying 20 μl of a 5-mg/ml solution in sterile water) in the absence of cAMP on SC medium (zone of inhibition [ZI], 51 ± 1 mm). Addition of 0.1 mM cAMP to the medium restored resistance to fluconazole to wild-type levels (ZI, 38 ± 1 mm [Table 1]). Higher concentrations of cAMP (1 mM) did not increase the resistance to fluconazole appreciably (ZI, 36 ± 1 mm). Similarly, the sensitivity of SR607 (wild type) to fluconazole was not affected significantly by the addition of 1 mM cAMP (ZI, 36 ± 1 and 34 ± 1 mm respectively [Table 1]). The plates were grown at 30°C for 24 h. Experiments were done in triplicate at different time points. These results could be interpreted as evidence that cAMP is required for activation of response mechanisms to protect the cell from fluconazole. The lack of a significantly increased resistance in the wild type by increased amounts of exogenous cAMP can be explained by the known resistance of wild-type strains to external cAMP and by the high internal levels of cAMP in wild-type cells (11).

TABLE 1.

Effects of cAMP and activated Ras on fluconazole resistance in S. cerevisiae^a

Strain	Mean ZI ± SD (mm) with cAMP at the following concn:
	0 mM	0.1 mM	1 mM
SR959b	51 ± 1	38 ± 1	36 ± 1
SR607b	36 ± 1	36 ± 1	34 ± 1
10560-14C/URA3c	38 ± 1
10560-14C/URA3 Ras2Val19c	33 ± 2
DK13-5D/URA3c	52 ± 1
DK13-5D/URA3 Ras2Val19c	44 ± 2

^aResistance tested using 100 μg of fluconazole by applying 20 μl of a 5-mg/ml fluconazole solution in sterile water to a paper disk. All experiments were performed in triplicate. 

^bApproximately 10⁵ cells were spread on SC plates, and growth was examined after 24 h at 30°C. 

^cApproximately 10⁵ cells were spread on SC-uracil plates, and growth was examined after 24 h at 30°C. 

Expression of RAS2^Val19 results in resistance to fluconazole in a PDR5-independent fashion.

RAS2^Val19 is a well-characterized dominant active allele of RAS2 (10). We transformed the Saccharomyces strain 10560-14C (MATa ura3-52 leu2::hisG his3::hisG [supplied by the Fink laboratory]) and DK 13-5D (MATa pdr5::Tn3::LEU2::lacZ ura3-52::leu2::hisG [6]) with either a single-copy plasmid containing URA3 as a selection marker and the allele RAS2^Val19 (S. Rupp, unpublished data) or with a URA3 plasmid containing no insert (pRS316 [8]). We selected the corresponding Ura⁺ transformants (10560-14C/URA3, pdr5/URA3, 10560-14C/URA3-RAS2^Val19, and pdr5/URA3-RAS2^Val19) in SC-uracil plates. To compare the fluconazole sensitivities of 10560-14C and DK13-5D with those of the respective isogenic strains expressing RAS2^Val19, the resulting strains were inoculated in 5 ml of liquid SC-uracil medium. Cultures were grown to saturation at 30°C, diluted (1:1,000) in 10 ml of SC-uracil medium, and grown at 30°C into late log phase. Yeast growth was examined by spreading approximately 10⁵ cells of each culture on SC-uracil plates containing 100 μg of fluconazole in a paper disk. Growth on plates at 30°C was evaluated for 24 h. Experiments were performed in triplicate at different time points. As shown in Table 1, the expression of RAS2^Val19 resulted in a small increase in fluconazole resistance in both the 10560-14C/RAS2^Val19 (ZI, 38 ± 1 mm in 10560-14C; ZI, 33 ± 2 mm in 10560-14C/RAS2^Val19) and in DK13-5D/Ras2^Val19 strains respectively (ZI, 52 ± 1 mm in DK13-5D; ZI, 44 ± 2 mm in DK13-5D/Ras2^Val19). The observed small protective effect in strains expressing Ras2^Val19 was not specific to fluconazole, because protection from cycloheximide (5 μg in a paper disk) was also seen in these strains on SC-uracil plates. On the other hand, toxicity from 5-fluorocytosine (50 μg in a paper disk) was no different in the strains expressing Ras2^Val19 (data not shown). Cycloheximide but not 5-fluorouracil is a substrate of Pdr5p (2).

In conclusion, our data suggest that cAMP is required for resistance to fluconazole. This is in agreement with our finding that the activation of the Ras pathway exerts some protective effect against fluconazole toxicity in Saccharomyces. The exact mechanism of protection is not known. However, cAMP and the Ras pathway may regulate either directly or indirectly the major facilitator superfamily network of transporters and thus the efficiency of drug efflux in Saccharomyces. This hypothesis is supported by the finding that CDC25, an upstream regulator of RAS2, affects the activity of the major facilitator superfamily glucose transporters (9). Elucidation of the role of cAMP in azole resistance could shed light on the mechanisms of resistance of the inherently resistant pathogenic fungi.