SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus

Abstract

Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP-mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Figure 1. SrbA regulates genes encoding enzymes involved in ergosterol biosynthesis and iron metabolism in response to hypoxia.

(A) Heat map representation of gene transcripts involved in ergosterol biosynthesis that are affected by A. fumigatus SrbA (B) Heat map representation of gene transcripts involved in iron metabolism that are affected by SrbA. A complete list of differentially expressed genes is available in Tables S1, S2, S3, S4. Data compare wild-type A. fumigatus to the ΔsrbA strain at the indicated times after exposure to hypoxic conditions, such that wild-type transcript levels after one hour hypoxia exposure is compared to ΔsrbA after one hour. Red indicates transcript levels are higher in ΔsrbA (fold changes are log base 2). Three biological replicates each with dye flips were performed for each time point examined.

Figure 2. qRT-PCR confirmation of SrbA-dependent iron homeostasis gene transcript abundance in hypoxia.

Transcript levels of fetC, ftrA, sidA, and sit1 were examined in normoxia then after a shift to hypoxia for 1, 2, and 4 hours in wild-type CEA10 and ΔsrbA strains. Transcript levels were normalized to β-tubulin transcript levels in each sample and data is presented relative to the wild-type transcript levels at time 0 in normoxia for each transcript using 2∧-ΔΔC_t method. Normalized fold expression less than 1 indicate the transcript levels are reduced in ΔsrbA compared to wild-type.

Figure 3. Enrichment of SrbA at the promoters of ergosterol biosynthesis and iron uptake genes.

Chromatin immunoprecipitation (ChIP) qPCR was performed on DNA from wild-type and ΔsrbA cells that were incubated in hypoxia for 4 hours. DNA was precipitated with either control IgG or 1 µg of anti-SrbA polyclonal antibody. Binding of SrbA to putative promoter regions was assessed with qPCR and data is presented as the percent enrichment of each sample to the input control. Results are the mean and standard deviation of 2 biological ChIP replicates and two qPCR technical replicates.

Figure 4. SrbA activates extracellular and intracellular siderophore production independent of SreA and HapX.

(A) Extracellular and (B) intracellular. Quantification of extracellular TAFC and intracellular FC and after growth for 24 hours at 37°C during iron-replete (+Fe) and depleted (−Fe) conditions was normalized to the biomass of the respective strain and furthermore to that of the CEA10 during iron starvation. The given values are the mean ± SD of six biological replicates. Under iron-replete conditions the measured FC was iron-free (desferri-FC), while under iron-replete conditions the FC was iron loaded (ferri-FC).

Figure 5. srbA expression is transcriptionally upregulated by iron starvation and SrbA-deficiency downregulates the SreA regulon independent of SreA and HapX, as well as the ergosterol biosynthetic erg3 and erg25 and the heme-biosynthetic hem13.

For Northern analysis, total RNA was isolated from A. fumigatus strains grown for 24 h in liquid cultures at 37°C at 200 rpm during iron-replete (+Fe) and depleted (−Fe) conditions. Ethidium bromide-stained rRNA is shown as control for loading and quality of RNA.

Figure 6. Increased iron availability and/or inactivation of SreA increase resistance of ΔsrbA to fluconazole.

E-test strips (AB Biodisk, bioMérieux) impregnated with a gradient of fluconazole were placed onto a MM agar plates representing different iron availability (−Fe; +Fe, 30 µM; hFe,10 mM iron) and containing a lawn of conidia. Growth inhibition was measured after 48 h at 37°C by direct observation.

Figure 7. Increased iron levels increase erg11A transcript and total ergosterol levels in the absence of SrbA.

qRT-PCR analysis of erg11A, erg11B, srbA, and erg25A transcript levels were measured in the wild-type CEA10 (A) and ΔsrbA strains (B). Transcript levels were normalized to tefA transcript levels in each sample and data was normalized to the −Fe sample in both strains examined. Chelator = 100 µM of the iron chelator 2,2-dipyridyl was added to the culture medium to completely remove free iron. Data represents the mean and standard deviation of three biological and two PCR technical replicates. (C) Total ergosterol content of respective strains in response to iron depleted and high iron conditions. Data represent the mean and standard deviation of 2 biological replicates. *,**, *** = p<0.05, two-tailed paired t-Test. *** refers to statistical comparisons between CEA10 in both Fe+ and Fe− to ΔsrbA in both Fe+ and Fe−.

Figure 8. Increased iron availability and/or inactivation of SreA improve growth of ΔsrbA during hypoxia.

For plate growth assays of CEA10, ΔsreA, ΔhapX, ΔsrbA, ΔsrbAΔsreA, and ΔsrbAΔhapX under normoxic and hypoxic conditions, 100 conidia of each strain were point-inoculated on AMM agar plates containing different iron concentrations (−Fe; +Fe, 30 µM; hFe,1.5 mM) or the iron chelator BPS (−Fe, 100 µM BPS) and incubated at 37°C for 96 h during hypoxic conditions or normoxic conditions.

Figure 9. Inactivation of SreA in ΔsrbA does not restore fungal virulence.

(A) Lung histopathology of CD1 mice infected with respective A. fumigatus strains on day +4 after infection. Substantial fungal growth and tissue necrosis are observed in lungs of mice infected with wild-type CEA10. However, little to no fungal growth is observed in lungs of mice infected with either ΔsrbA, or ΔsrbAΔsreA. (B) Kaplan-Meier survival analysis of respective A. fumigatus strains in chemotherapeutic model of invasive pulmonary aspergillosis. As with previously published results with strain ΔsrbA, strain ΔsrbAΔsreA has a significant reduction in virulence compared to wild-type CEA10 (P<0.0001, Log-Rank Test for comparison between ΔsrbAΔsreA and wild-type CEA10,).

Figure 10. Genes involved in amino acid biosynthetic processes are transcriptionally affected by loss of SrbA in hypoxia.

Heat map representation of genes involved in amino acid biosynthesis that are regulated by A. fumigatus SrbA. A detailed list of genes and fold changes is available in Tables S1, S2, S3, S4. Data compare wild-type A. fumigatus to the ΔsrbA strain at the indicated times after exposure to hypoxic conditions, such that wild-type transcript levels after one hour hypoxia exposure is compared to ΔsrbA after one hour. Red indicates transcript levels are higher in ΔsrbA (fold changes are log base 2). Three biological replicates each with dye flips were performed for each time point examined.

Figure 11. Model for relationships between the transcriptional regulators SrbA, SreA, HapX, and AcuM and their roles in iron acquisition and ergosterol biosynthesis.