Abnormal Ergosterol Biosynthesis Activates Transcriptional Responses to Antifungal Azoles

Abstract

Fungi transcriptionally upregulate expression of azole efflux pumps and ergosterol biosynthesis pathway genes when exposed to antifungal agents that target ergosterol biosynthesis. To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol. Using an inducible promoter to regulate Neurospora crassa erg11, which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles. By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by cdr4 and, unexpectedly, genes encoding ergosterol biosynthesis enzymes (erg genes) that are responsive to azoles, are not dependent on ergosterol depletion. Nonetheless, deletion of erg2, which encodes C-8 sterol isomerase, also induced expression of cdr4. Deletion of erg2 also induced the expression of erg24, the gene encoding C-14 sterol reductase, but not other tested erg genes which were responsive to erg11 inactivation. This indicates that inhibition of specific steps of ergosterol biosynthesis can result in different transcriptional responses, which is further supported by our results obtained using different ergosterol biosynthesis inhibitors. Together with the sterol profiles, these results suggest that the transcriptional responses by cdr4 and erg genes are associated with accumulation of specific sterol intermediate(s). This was further supported by the fact that when the erg2 mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway. Even though cdr4 expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of cdr4 did not result in efflux of the accumulated intermediate(s). This study demonstrates, by detailed genetic and chemical analysis, that transcriptional responses by a major efflux pump and genes of the ergosterol biosynthesis pathway to ergosterol biosynthesis inhibitors can be independent of the presence of the drugs and are linked with the accumulation of ergosterol intermediate(s).

Keywords: C-8 sterol isomerase; azoles; efflux pump; sterol 14α-demethylase; sterol intermediate; stress response; tcu-1 promoter.

FIGURE 1

Suggested ergosterol biosynthesis pathway in N. crassa and transcriptional responses to disruption at certain steps. Each enzyme relates to one step of the ergosterol biosynthesis pathway in N. crassa. The most marked difference between the N. crassa and S. cerevisiae is the step catalyzed by ERG6 (marked by an asterisk). This step follows the reactions catalyzed by ERG25, ERG26 and ERG27 in yeast. Corresponding genes encoding enzymes in bold were analyzed in this study. Dotted arrows show the alternative biosynthesis pathways when the main pathway is impaired. Dotted lines showed the genes transcriptional induced by ergosterol biosynthesis inhibitors or erg gene deletion.

FIGURE 2

erg11 is an essential gene in N. crassa. (A) Schematic diagram of the process of Ptcu-1::erg11 (NCU02624, encoding sterol 14α-demethylase) strain construction. (B,C) erg11 is required for fungal growth. Conidia of N. crassa bd Ku70^RIP strain (the parental strain), Ptcu-1::erg11 mutant and the complemented Perg11::erg11 mutant were inoculated on Vogel’s plates (B) and Vogel’s slants (C) supplemented with or without CuSO₄ or Cu²⁺ chelator BCS and grown at 28°C for 7 days (B) or 30 h (C), respectively. (D) erg11 inactivation resulted in sterol content changes in N. crassa. After grown for 13.5 h in liquid Vogel’s medium amended with BCS, the mycelium was then transferred to liquid Vogel’s medium amended with BCS and CuSO₄ and treated for 22 h. The sterols were then extracted and subjected to HPLC-MS analysis. The abundance of sterols was calculated on the basis of the chromatogram peak area and normalized using an internal control and sample weight. The results presented here are means of two biological replicates. (E) Conidia of bd Ku70^RIP and Ptcu-1::erg11 were germinated in Vogel’s minimal medium supplemented with BCS or CuSO₄ for 10 h. Hyphae were imaged after fixing and staining with calcofluor white and DAPI. Clustered nuclei are indicated by arrows and enlarged (below panel on the right).

FIGURE 3

Transcriptional induction of cdr4 and erg genes following erg11 inactivation. (A) Detection of ERG11 (sterol 14α-demethylase) by Western Blotting in the Ptcu-1::erg11 strain grown in Vogel’s medium supplemented with Cu²⁺ or BCS using anti-myc antibodies. The parental strain (bd Ku70^RIP) grown in Vogel’s medium supplemented with BCS and the Ptcu-1::erg11 strain grown in normal Vogel’s medium was used as controls. (B) After grown in medium containing BCS for 13.5 h, mycelium were then shifted to indicated conditions to grow for additional 22 h. The transcript levels of selected genes were examined in the N. crassa bd Ku70^RIP strain and Ptcu-1::erg11 strain treated with 50 μM BCS and 100 μM CuSO₄ for 22 h. Transcript levels of cdr4 (NCU05591, encoding azole efflux pump CDR4), erg5 (NCU05278, encoding C-22 sterol desaturase), erg6 (NCU03006, encoding sterol C-24 methyl transferase), erg11 (NCU02624, encoding sterol 14α-demethylase) and erg24 (NCU08762, encoding C-14 sterol reductase) were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression was calculated by 2^-ΔΔCt method and normalized to β-tubulin. The results presented here are means of three biological replicates, and the significant levels between the two strains in each treatment were calculated by t-test and marked as ^∗(p < 0.05), ^∗∗(p < 0.01) or ^∗∗∗(p < 0.001).

FIGURE 4

Phenotypic analysis and gene expression analysis of ergosterol deficient mutants. Conidia of N. crassa wild type, Δerg2 (NCU04156, encoding C-8 sterol isomerase), Δerg3 (NCU06207, encoding C-5 sterol desaturase), Δerg4 (NCU01333, encoding C-24(28) sterol reductase), and Δerg5 (NCU05278, encoding C-22 sterol desaturase) mutants were inoculated on Vogel’s slants (A) and Vogel’s plates (C) and grown at 28°C for 7 days (A) or 30 h (C), respectively. (B) Major sterols in ergosterol deficient mutants. Mycelium grown in liquid Vogel’s medium for 36 h was used for sterol extraction. The extracted sterols were then subjected to HPLC-MS analysis. Ergosterol was identified by UV absorbance at 280 nm and referred to an ergosterol standard. Other sterols accumulated were further checked by MS and listed in Supplementary Table 4. The sterols marked with numbers are ergosterol for 1, ergosta-5,7,22,24(28)-tetraenol for 2, ergosta-5,7,24(28)-trienol for 3, ergosta-5,7-dienol for 4. (D) Transcript levels of cdr4 (NCU05591, encoding azole efflux pump CDR4), erg2, erg5, erg6 (NCU03006, encoding sterol C-24 methyl transferase), erg11 (NCU02624, encoding sterol 14α-demethylase) and erg24 (NCU08762, encoding C-14 sterol reductase) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in the selected mutants. The expression was calculated by 2^-ΔΔCt method and normalized to β-tubulin. The results presented here are means of three biological replicates, and the significant levels of cdr4 and erg24 between WT and Δerg2 were calculated by t-test and marked as ^∗(p < 0.05), ^∗∗(p < 0.01) or ^∗∗∗(p < 0.001).

FIGURE 5

Differential transcriptional responses to different ergosterol biosynthesis inhibitors. After grown in liquid media for 13.5 h, the N. crassa wild-type strain were treated with 2 mg/L ketoconazole and 0.375 mg/L Amorolfine for 22 h, same amount dissolvent treatment was used as a control. Transcript levels of cdr4 (NCU05591, encoding azole efflux pump CDR4), erg2 (NCU04156, encoding C-8 sterol isomerase), erg5 (NCU05278, encoding C-22 sterol desaturase), erg6 (NCU03006, encoding sterol C-24 methyl transferase), erg11 (NCU02624, encoding sterol 14α-demethylase) and erg24 (NCU08762, encoding C-14 sterol reductase) were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression was calculated by the 2^-ΔΔCt method and normalized to β-tubulin. The results presented here are means of three biological replicates. The significant levels were calculated by t-test and marked as ^∗(p < 0.05), ^∗∗(p < 0.01) or ^∗∗∗(p < 0.001).

FIGURE 6

ERG11 inhibition overrides the effects of erg2 deletion. After grown in liquid media for 13.5 h, the N. crassa wild-type and Δerg2 strains were treated with 2 mg/L ketoconazole for 22 h and same amount DMSO was used as a control. Transcript levels of cdr4 (NCU05591, encoding azole efflux pump CDR4), erg5 (NCU05278, encoding C-22 sterol desaturase), erg6 (NCU03006, encoding sterol C-24 methyl transferase), erg11 (NCU02624, encoding sterol 14α-demethylase) and erg24 (NCU08762, encoding C-14 sterol reductase) were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression was calculated by 2^-ΔΔCt method and normalized to β-tubulin. The results presented here are means of three biological replicates. The significant levels were calculated by t-test and marked as ^∗(p < 0.05), ^∗∗(p < 0.01), or ^∗∗∗(p < 0.001).

FIGURE 7

Transcriptional response of cdr4 and erg11 to 2 mg/L ketoconazole (KTC). After grown in liquid media for 13.5 h, KTC was added and grew for another 24 h. The gene expression of cdr4 (NCU05591, encoding azole efflux pump CDR4) and erg11 (NCU02624, encoding sterol 14α-demethylase) were monitored at various time points following KTC treatment. Transcript levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression was calculated with 2^-ΔΔCt method and normalized to β-tubulin.

FIGURE 8

Sterol content between the N. crassa wild-type and Δcdr4 strain. After grown in liquid media for 13.5 h, the N. crassa wild-type and Δcdr4 strains were treated with 2 mg/L ketoconazole for 22 h and same amount DMSO was used as a control. KTC (A) and Sterols (B) were then extracted and measured by HPLC-MS. The abundance of sterols was calculated on the basis of the chromatogram peak area and normalized using an internal control and sample weight. The results presented here are means of three biological replicates. The significant levels were calculated by t-test and marked as ^∗(p < 0.05), ^∗∗(p < 0.01), or ^∗∗∗(p < 0.001).