Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors

Abstract

Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation of ERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candida species. RNA analysis revealed that ERG11 expression in C. albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two- to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth. ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal ERG11 upregulation was reversed. Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly, ERG11 upregulation occurred following treatment of C. albicans cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for global ERG upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C. tropicalis, C. glabrata, and C. krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

FIG. 1

Ergosterol biosynthesis pathway. Only selected substrates or products are shown. Genes (in italics) that encode enzymes in the pathway are labeled according to the convention for S. cerevisiae; those whose expression was monitored in this study are shown in bold. The three groups of sterol biosynthesis inhibitors examined in this study are underlined and are shown to the right of the gene encoding the targeted enzyme. CoA, coenzyme A.

FIG. 2

Effect of azole treatment on ERG11 expression in C. albicans. (A) RNA slot blot analysis of ACT1, ERG11, and CDR1 expression in C. albicans 24433 following fluconazole treatment (0, 2, or 8 μg/ml) for the indicated times. Similar results were observed in four independent experiments and with a second C. albicans strain (strain ATCC 90028). (B) RNA slot blot analysis of ACT1 and ERG11 expression in C. albicans 24433 following treatment with the indicated azoles for the indicated times. Azole concentrations were 9 μg/ml (fluconazole) and 0.5 μg/ml (all others). The control received DMSO vehicle (final concentration, 0.25%). Similar results were observed in a second independent experiment.

FIG. 3

Correlation of ERG11 expression and C. albicans 24433 growth in control and fluconazole-treated cultures. RNA levels (determined from slot blot analysis and normalized to ACT1 levels) are represented as fold increase or decrease relative to the level for the control at 0 h; solid bars, 0 μg of fluconazole per ml; open bars, 9 μg of fluconazole per ml. C. albicans growth is represented as cell number (determined in a hemocytometer) at the indicated times and treatments: no drug (■) and 9 μg of fluconazole per ml (▵). The data shown represent the averages of three independent experiments.

FIG. 4

Effects of medium and pH on fluconazole-dependent ERG11 upregulation in C. albicans 24433. (A) Media were YPD, YPD supplemented with 20% fetal bovine serum (Serum), RPMI 1640 medium prepared according to the susceptibility testing guidelines in document M27-A (26) (RPMI), and supplemented yeast nitrogen base (DOB). Effects on ERG11 expression were analyzed by slot blot analysis with normalization to ACT1 RNA levels and are represented as the fold change relative to the level for the untreated controls (solid bars). Treatment was for 2 h with fluconazole at 1 (hatched bars) or 9 (open bars) μg/ml. The data shown are the averages of two independent experiments. (B) RPMI 1640 medium was adjusted to the indicated pH, and ERG11 expression was analyzed by RT-PCR. Incubation was at 35°C instead of 30°C to conform with the guidelines in document M27-A (26). Treatment was for 0, 3, or 5 h, as indicated, with no drug (solid bars) or fluconazole at 9 μg/ml (open bars). The data represent the averages of four RT-PCRs from two independent experiments.

FIG. 5

Global upregulation of C. albicans ERG genes following fluconazole treatment. RT-PCR was used to analyze the relative expression of ACT1, CDR (CDR1 and CDR2), and the indicated ERG genes in cultures (strain 24433) treated with fluconazole at 9 μg/ml (labeled 9) or no drug (labeled 0) for 0, 1, 3, or 5 h, as indicated. Equal volumes from cDNA reactions with (+) or without (−) reverse transcriptase (RT) were amplified in parallel to detect genomic DNA contamination. Lane G, PCR positive control containing genomic DNA; lane N, PCR negative control without added template. The data shown are representative of two independent experiments, and similar data were obtained with an additional C. albicans isolate (isolate 630-15.3). Due to differences in basal levels of expression, PCR cycle numbers were optimized for each gene to ensure logarithmic-phase amplification. The cycle numbers used were 23 (ERG11 and ACT1), 25 (CDR), 28 (ERG3, ERG7, ERG9), and 30 (ERG1 and ERG25). On the basis of previous slot blot data, CDR expression is primarily due to CDR1.

FIG. 6

Upregulation of C. albicans ERG11 following treatment with terbinafine or fenpropimorph. ERG11 RNA levels were examined by slot blot hybridization (terbinafine) or RT-PCR (fenpropimorph), normalized to ACT1 RNA levels, and represented as the fold change relative to the RNA levels at 0 h. Cultures were treated with no drug (solid bars) or the indicated drug at 1 (hatched bars) and 9 (open bars) μg/ml for the indicated times.

FIG. 7

Upregulation of ERG11 in three additional Candida species following azole treatment. RNA levels were examined by RT-PCR after 0, 3, and 5 h of treatment, as indicated, and are represented as the fold change relative to the levels at 0 h. Cultures were treated with no drug (solid bars), fluconazole at 9 μg/ml (hatched bars), or itraconazole at 0.1 μg/ml (open bars).