The metalloreductase FreB is involved in adaptation of Aspergillus fumigatus to iron starvation

Abstract

Aspergillus fumigatus employs two high affinity iron uptake mechanisms, siderophore mediated iron uptake and reductive iron assimilation (RIA). The A. fumigatus genome encodes 15 putative metalloreductases (MR) but the ferrireductases involved in RIA remained elusive so far. Expression of the MR FreB was found to be transcriptionally repressed by iron via SreA, a repressor of iron acquisition during iron sufficiency, indicating a role in iron metabolism. FreB-inactivation by gene deletion was phenotypically largely inconspicuous unless combined with inactivation of the siderophore system, which then decreased growth rate, surface ferrireductase activity and oxidative stress resistance during iron starvation. This study also revealed that loss of copper-independent siderophore-mediated iron uptake increases sensitivity of A. fumigatus to copper starvation due to copper-dependence of RIA.

Supplementary Fig. S1

Generation of the A. fumigatus strains ΔfreB, ΔfreBΔsidA, and freB^cΔsidA and respective southern blot analysis. The freB deletion was realized using the bipartite marker technique. A. fumigatus wt and ΔsidA strains were co-transformed with two DNA fragments, each containing overlapping, but incomplete fragments of the pyrithiamine resistance-conferring ptrA gene fused to the freB 5′- and 3′-flanking sequences (1.0 kb and 0.8 kb), respectively. Deletion freB (B) resulted from a homologous recombination between the wt locus (A) and the transformation constructs by a triple cross over of flanking regions and within the marker cassette. This strategy replaced the freB region +230 to +2356 (according to the transcription start site) with the ptrA gene. For the reconstitution of freB in ΔfreBΔsidA. A 5649-bp PCR fragment generated with primers oFreB7 and oFreB5r was used for co-transformation with the plasmid pAN8.1. freB^cΔsidA resulted from homologous recombination (single cross over) between the PCR amplified freB locus and the freB mutant allele (C). Southern blot analysis of PstI-digested genomic DNA of wt, ΔfreB, ΔsidA, ΔfreBΔsidA and freB^cΔsidA hybridized with the freB 5′-flanking region amplified with primers oFreB1 and oFreB3r is shown in (D).

Fig. 1

Phylogentic analysis (rooted neighbor joining tree) of MR from 5 asomycetous and two basidiomycetous species. For MR previously analyzed functionally or transcriptionally (see Section 3.1) the protein names are given. Abbreviations are the following: AFUA. A. fumigatus; AN. A. nidulans; Ca. C. albicans; CNAG. C. neoformans; Sc. S. cerevisiae; SPBC. S. pombe; UM. U. maydis.

Fig. 2

Expression of freB is repressed by iron via SreA but not by copper (A) and copper starvation transcriptionally upregulates high-affinity iron uptake in ΔfreBΔsidA (B). For Northern analysis A. fumigatus strains were grown for 24 h in liquid flask cultures under iron/copper-replete (++), iron depleted (−Fe), and copper depleted (−Cu) conditions. The SreA target genes sidA, mirB, and fetC encode the siderophore-biosynthetic ornithine monooxygenase, a siderophore transporter, and the RIA component ferroxidase, respectively (Haas et al., 2008). Ethidium bromide-stained rRNA is shown as control for loading and quality of RNA.

Fig. 3

SidA-deficiency results in sensitivity to starvation for copper and iron, respectively, (A) and FreB-deficiency results in the inability of colony formation from single conidia during iron starvation (B). (A) 10⁴ conidia of the respective strain were point-inoculated on AMM plates containing the different iron or copper concentrations (+Fe, 30 μM; hFe, 1.5 mM; + Cu, 16 μM) and were incubated for 48 h at 37 °C. (B) About 20 conidia were plated on copper-replete media containing the indicated iron concentration. BPS inhibits strains lacking the siderophore system by blocking of RIA (Schrettl et al., 2004). Copper starvation blocks formation of the green conidial pigmentation resulting in yellow pigmentation.

Fig. 4

FreB-deficiency reduces ferrireductase activity (A), increases sensitivity to hydrogen peroxide (B), and increases cellular reactive oxygen species in particular in the absence of siderophore biosynthesis (ΔfreBΔsidA). Ferrireductase activity was measured after 24 h of submersed growth at 37 °C during iron starvation and normalized to that of the wt. Hydrogen peroxide sensitivity was analyzed after 48 h of growth in plate diffusion assays. The level of cellular reactive oxygen species was quantified without (open bars) and with (grey bars) hydrogen peroxide treatment by the dichlorofluorescein assay, in which fluorescence is proportional to the level of reactive oxygen species. The given values are the mean ± STD of three biological replicates. Statistical significance was analyzed as described in Table 2 (^*p < 0.05; ^**p < 0.01).