SREBP-dependent triazole susceptibility in Aspergillus fumigatus is mediated through direct transcriptional regulation of erg11A (cyp51A)

Abstract

As triazole antifungal drug resistance during invasive Aspergillus fumigatus infection has become more prevalent, the need to understand mechanisms of resistance in A. fumigatus has increased. The presence of two erg11 (cyp51) genes in Aspergillus spp. is hypothesized to account for the inherent resistance of this mold to the triazole fluconazole (FLC). Recently, an A. fumigatus null mutant of a transcriptional regulator in the sterol regulatory element binding protein (SREBP) family, the ΔsrbA strain, was found to have increased susceptibility to FLC and voriconazole (VCZ). In this study, we examined the mechanism engendering the observed increase in A. fumigatus triazole susceptibility in the absence of SrbA. We observed a significant reduction in the erg11A transcript in the ΔsrbA strain in response to FLC and VCZ. Transcript levels of erg11B were also reduced but not to the extent of erg11A. Interestingly, erg11A transcript levels increased upon extended VCZ, but not FLC, exposure. Construction of an erg11A conditional expression strain in the ΔsrbA strain was able to restore erg11A transcript levels and, consequently, wild-type MICs to the triazole FLC. The VCZ MIC was also partially restored upon increased erg11A transcript levels; however, total ergosterol levels remained significantly reduced compared to those of the wild type. Induction of the erg11A conditional strain did not restore the hypoxia growth defect of the ΔsrbA strain. Taken together, our results demonstrate a critical role for SrbA-mediated regulation of ergosterol biosynthesis and triazole drug interactions in A. fumigatus that may have clinical importance.

Fig 1

FLC and VCZ increase erg11A transcript levels. (A) CBS 144.89 germlings were treated with FLC (128 μg/ml) for 6 or 12 h at 37°C with agitation. (B) CBS 144.89 germlings were treated with VCZ (0.0625 μg/ml) for 6 or 12 h at 37°C with agitation. qRT-PCR was conducted to determine the effects of these triazoles on erg11A and erg11B mRNA abundance. Data represent three biological and two combined technical replicates. mRNA abundance was normalized to the housekeeping genes tefA and actin and are relative to CBS 144.89. ψ, P < 0.05; *, P < 0.01.

Fig 2

Differential erg11A and erg11B transcript abundance in the ΔsrbA strain compared to that in CBS 144.89. RNA samples were extracted from each respective strain 12 h postgermination (in liquid GMM) at 37°C, 300 rpm. mRNA abundance was normalized to the housekeeping genes tefA and actin and are relative to CBS 144.89. Results are the means and standard deviations of two biological replicates for CBS 144.89 and three biological replicates for the ΔsrbA strain. ψ, P < 0.05; n.s., not significant.

Fig 3

srbA is required for transcriptional responses to FLC and VCZ. (A) ΔsrbA germlings were treated with FLC (0.5 μg/ml, 50% less than the observed MIC) for 6 or 12 h at 37°C with agitation. (B) ΔsrbA germlings were treated with VCZ (0.006 μg/ml, 50% less than the observed MIC) for 6 or 12 h at 37°C with agitation. qRT-PCR was conducted to determine the effect of these triazoles on erg11A and erg11B mRNA abundance. Data represent three biological and two combined technical replicates. mRNA abundance was normalized to the housekeeping genes tefA and actin and are relative to CBS 144.89. ψ, P <0.05; *, P < 0.001; ns, not significant.

Fig 4

Generation of erg11A inducible strain in ΔsrbA background. (A) Locus of erg11A locus in the CBS 144.89 (wild-type) strain. (B) Locus of the pniiA-erg11A construct in the ΔsrbA background strain. (C) Southern blot analysis of the wild-type and mutant strains. XbaI/XmaI genomic DNA was digested for 18 h at 37°C. Strains were separated on a 1% agarose gel, transferred, and hybridized with a 467-bp probe. (D) Induction/repression of the pniiA-erg11A-ΔsrbA strain correlates with mRNA abundance of erg11A (P = 0.008). RNA samples were extracted from each respective strain 12 h postgermination in liquid GMM plus 20 mM NO₃ (CBS 144.89, ΔsrbA, pniiA-erg11A-ΔsrbA induced [ind]) or liquid GMM plus 20 mM NH₄ (pniiA-erg11A-ΔsrbA repressed [rep]) at 37°C, 300 rpm. mRNA abundance was normalized to the housekeeping genes tefA and gadpH and are relative to CBS 144.89. Results are the means and standard deviations of three biological replicates.

Fig 5

Restoration of erg11A transcripts restores FLC and VCZ MICs in the ΔsrbA strain. Etest strip concentrations (in micrograms per milliliter) for A. fumigatus CBS 144.89, the ΔsrbA strain, and pniiA-erg11A-ΔsrbA strain. A clear ellipse indicates susceptibility to FLC or VCZ. (A) As expected, FLC has no effect on CBS 144.89, and the ΔsrbA strain is susceptible to FLC (MIC, 1.0 μg/ml). Wild-type expression of the pniiA-erg11A-ΔsrbA strain reconstitutes FLC resistance. Likewise, repression of the pniiA-erg11A-ΔsrbA strain restores FLC susceptibility as observed in the ΔsrbA strain. (B) erg11A transcript expression partially restores the VCZ MIC in the ΔsrbA strain. CBS 144.89 MIC, 0.19 (induced or repressed); ΔsrbA strain MIC, 0.008 (induced or repressed); pniiA-erg11A-ΔsrbA strain MICs, 0.064 (induced) and 0.008 (repressed).

Fig 6

SrbA directly binds erg11A promoter region sequence in vitro. Electrophoretic mobility shift assays were conducted utilizing a LightShift chemiluminescent EMSA kit from Thermo Scientific. A total of 40 fmol of a 118-bp biotin-labeled probe from erg11A upstream region and 1 μg purified SrbA protein were coincubated at room temperature for 20 min. Recombinant polyclonal SrbA antibody was used for the supershift assay. Lane 1, no protein control; lane 2, EMSA reaction; lane 3, supershift assay.

Fig 7

Restoration of erg11A does not affect hyphal growth in normoxia or restore ΔsrbA-related hypoxic growth defect. A total of 10⁵ conidia was spotted on solid media (GMM plus 20 mM NO₃ [induction] or 20 mM NH₄ [repression]) under normoxic (N) or hypoxic (H) conditions. (A) Comparison of colony growth diameter on nitrate (NO₃) in both hypoxia (H) and normoxia (N). (B) Comparison of colony growth diameter on ammonium (NH₄) in both hypoxia (H) and normoxia (N). Values represent the means and standard deviations of 3 biological replicates. (C) Radial growth at 120 h.

Fig 8

Genetic deletion of erg11A (Δerg11A) or erg11B (pniiA-erg11A-Δerg11B) does not affect hyphal growth in normoxia or hypoxia. A total of 10⁵ conidia was spotted on solid media (GMM plus 20 mM NO₃ [induction] or 20 mM NH₄ [repression]) under normoxic or hypoxic conditions. (A) Radial growth at 96 h. (B) Comparison of colony growth diameter on nitrate (NO₃) in both hypoxia and normoxia. (C) Comparison of colony growth diameter on ammonium (NH₄) in both hypoxia and normoxia. Values represent the means and standard deviations of 3 biological replicates.

Fig 9

Total ergosterol is unchanged by restoration of erg11A transcript levels in the ΔsrbA strain. Total ergosterol from A. fumigatus strains was extracted 12 h postgermination in liquid GMM with 20 mM NO₃ (pniiA-erg11A-ΔsrbA ind, ΔsrbA, and CBS 144.89) or liquid GMM with 20 mM NH4 (pniiA-erg11A-ΔsrbA rep). Mycelia were harvested via vacuum filtration and then dried and weighed. Two hundred milligrams of dry fungal tissue was extracted as described previously (3). Upon preparation for injection, samples were resolved in 1 ml of methanol and syringe filtered. Total ergosterol was measured using a Shimadzu CLASS-VP HPLC and detected via a SPD-M10AVP PDA at 280 nm on a μBondapack C₁₈ column (300 mm by 3.9 mm, 10 μm). *, P < 0.001. Values represent the means and standard deviations of 3 to 5 biological replicates and three technical replicates.

Fig 10

Sterol intermediates demonstrate a blockage at Erg25 in the ΔsrbA strain. Ratio of sterol intermediates to ergosterol. Sterols were measured via GC-MS using a Shimadzu QP-2010 GC with a quadrupole mass ionizer. Peaks were identified via EI fragmentation patterns. A, 4,24-dimethyl cholesta-8,24(28)-dien-3β-ol; B, 4,4,24-trimethyl cholesta-8,24(28)-trien-3β-ol; and C, 4,14,24-trimethyl ergosta-8, 24(28)-dien-3β-ol.