Regulation of High-Affinity Iron Acquisition, Including Acquisition Mediated by the Iron Permease FtrA, Is Coordinated by AtrR, SrbA, and SreA in Aspergillus fumigatus

Abstract

Iron acquisition is crucial for virulence of the human pathogen Aspergillus fumigatus. Previous studies indicated that this mold regulates iron uptake via both siderophores and reductive iron assimilation by the GATA factor SreA and the SREBP regulator SrbA. Here, characterization of loss of function as well as hyperactive alleles revealed that transcriptional activation of iron uptake depends additionally on the Zn₂Cys₆ regulator AtrR, most likely via cooperation with SrbA. Mutational analysis of the promoter of the iron permease-encoding ftrA gene identified a 210-bp sequence, which is both essential and sufficient to impart iron regulation. Further studies located functional sequences, densely packed within 75 bp, that largely resemble binding motifs for SrbA, SreA, and AtrR. The latter, confirmed by chromatin immunoprecipitation (ChIP) analysis, is the first one not fully matching the 5'-CGGN₁₂CCG-3' consensus sequence. The results presented here emphasize for the first time the direct involvement of SrbA, AtrR, and SreA in iron regulation. The essential role of both AtrR and SrbA in activation of iron acquisition underlines the coordination of iron homeostasis with biosynthesis of ergosterol and heme as well as adaptation to hypoxia. The rationale is most likely the iron dependence of these pathways along with the enzymatic link of biosynthesis of ergosterol and siderophores. IMPORTANCE Aspergillus fumigatus is the most common filamentous fungal pathogen infecting humans. Iron acquisition via siderophores has previously been shown to be essential for virulence of this mold species. Here, we demonstrate that AtrR, a transcription factor previously shown to control ergosterol biosynthesis, azole resistance, and adaptation to hypoxia, is essential for activation of iron acquisition, including siderophore biosynthesis and uptake. Dissection of an iron-regulated promoter identified binding motifs for AtrR and the two previously identified regulators of iron acquisition, SrbA and SreA. Altogether, this study identified a new regulator required for maintenance of iron homeostasis, revealed insights into promoter architecture for iron regulation, and emphasized the coordinated regulation of iron homeostasis ergosterol biosynthesis and adaptation to hypoxia.

Keywords: Aspergillus fumigatus; fungi; iron; molds; regulation; siderophore; siderophores; transcription factor.

FIG 1

Loss of AtrR causes an iron-dependent growth defect in A. fumigatus. To determine the role of AtrR in iron adaptation, 1 × 10⁴ conidia of wt (Afs35), ΔatrR, ΔsrbA, atrR^3X-HA, and atrR^phpsA ΔsrbA strains were point inoculated on AMM agar plates containing different iron concentrations (−Fe, without iron addition) or the iron chelator BPS (0.2 mM). The plates were incubated at 37°C for 48 h under normoxic or hypoxic (0.2% O₂) conditions.

FIG 2

Similar to SrbA, AtrR plays a crucial role in liquid growth and siderophore biosynthesis during iron starvation. (A) For determination of biomass production, 1 × 10⁶ conidia/mL were inoculated in 100 mL liquid AMM containing different iron concentrations or lacking iron supplementation (−Fe) and shaken at 200 rpm for 24 h at 37°C. Biomass was normalized to the wt grown under the same condition (0.194 ± 0.01 for −Fe, 0.556 ± 0 with 0.001 mM Fe, 0.659 ± 0.03 with 0.03 mM Fe, and 0.655 ± 0.02 with 5 mM Fe). Production of extracellular TAFC (B) and intracellular FC (C) was quantified after growth for 24 h at 37°C in −Fe and 0.001 mM Fe. Siderophore production was normalized to the respective biomass and subsequently to that of the wt grown under the same condition. The values are means and standard deviations (SD) for biological triplicates. *, P ≤ 0.001 relative to the wt according to two-way analysis of variance (ANOVA).

FIG 3

Similar to SrbA, AtrR is crucial for transcriptional activation of genes involved in adaptation to iron starvation as well as biosynthesis of heme and ergosterol. For Northern blot analysis, total RNA was isolated from A. fumigatus strains grown during iron limitation (−Fe) or iron sufficiency (+Fe) for 24 h at 37°C. Ethidium bromide-stained rRNA is shown as a control for loading and quality of RNA. Genes are described in the text and Table S2.

FIG 4

Nucleotide sequence of the bidirectional intergenic region of ftrA and fetC. The transcription start sites of both ftrA and fetC are in bold and underlined. The putative DNA binding motifs of different TFs are highlighted: SreA (5′-ATCWGATAA-3′ combined with a preceding GATAA motif on the complementary strand) in green, SrbA (5′-TCANNCCA-3′) in purple, AtrR (5′-CGG-X₁₂-CCG-3′) in red, and a putative SrbA binding motif overlapping the SreA motif in purple. The start sites of the different truncations and the deletions are indicated above the sequence by blue arrows and black lines, respectively. The nucleotide numbering refers to the ftrA TSS.

FIG 5

Truncations and deletion of pftrA using luc as a reporter for promoter activity identified functional regions. Promoter activity was measured during iron limitation (−Fe) and sufficiency (+Fe) as described in Materials and Methods. The promoter activities are means and SD for biological triplicates normalized to P1 (top) or P4 (bottom). The −Fe/+Fe column displays the ratio of promoter activity under −Fe and +Fe iron conditions of the different pftrA versions. Values in red and green indicate decreased and increased promoter activity relative to P1 or P4, respectively. The raw data are shown in Table S4A.

FIG 6

pftrA²¹⁰ mediates that iron regulation when fused with the pxylP²⁸⁹ minimal promoter. Promoter activity was measured as described in Materials and Methods. Data are means and SD for three biological replicates normalized to P3 grown under −Fe or +Fe conditions. The −Fe/+Fe column displays the ratio of promoter activity under −Fe and +Fe conditions of the different promoters. The raw data are shown in Table S4B.

FIG 7

Site-directed mutagenesis identified regulatory motifs in pftrA using luc as the reporter gene. Promoter activity was measured as described in Materials and Methods. Data are means and SD for three biological replicates normalized to P3 grown under −Fe or +Fe conditions. The −Fe/+Fe column displays the ratios of promoter activity under −Fe and +Fe iron conditions of the different pftrA versions. Values in red and green indicate decreased and increased promoter activity relative to P3, respectively. The raw data are shown in Table S4C.

FIG 8

ChIP analysis combined with luc promoter activity analysis confirms binding of AtrR to the predicted motif. The luc promoter activity was analyzed as described in Fig. 7 and normalized to P4. Data are means and SD of three biological replicates normalized to P4 grown during iron starvation (−Fe). The raw data are shown in Table S4A, ChIP was performed as described in Materials and Methods. Enrichment of AtrR at the P4 pftrA variants at the fcyB locus by ChIP during iron starvation (−Fe) was normalized to that at the native pftrA. The values are means and SD representing two independent ChIP reactions with three quantitative real-time PCRs from each ChIP experiment. Statistically significant differences from P4 according to an unpaired two-tailed t test are indicated: *, P < 0.01; **, P < 0.001.