New Results

Cooperative [2Fe-2S] cluster-binding regulates the functional transitions of the Aspergillus fumigatus iron regulator HapX for adaptation to iron starvation, sufficiency and excess

Simon Oberegger, Matthias Misslinger, Hubertus Haas
doi: https://doi.org/10.1101/2024.11.27.625597

ABSTRACT

Accurate sensing of cellular iron levels is vital, as this metal is essential but toxic in excess. The iron-sensing transcription factor HapX is crucial for virulence of Aspergillus fumigatus, the predominant human mold pathogen. Its absence impairs growth under iron limitation and excess, but not under moderate iron availability, suggesting that HapX switches between three states to adapt to varying iron availability.

This study suggests that the HapX state transitions are regulated by the different propensities of four phylogenetically conserved cysteine-rich regions (CRRs) to coordinate [2Fe-2S] clusters resulting in cumulative occupancies that depend on iron availability. In the iron starvation state, CRR-B and -C lack [2Fe-2S] clusters, the iron sufficiency/”neutral” state features clusters in CRR-B and/or -C and the iron excess state has clusters in all CRR-A, B, and -C, while CRR-D plays a minor role. Combinatorial mutation of CRR-B and -C blocked growth by locking HapX in the iron starvation state, leading to uncontrolled iron uptake, iron accumulation, repression of iron-consuming pathways and impaired iron detoxification. Loss of the C-terminal 27 amino acid region of HapX, which is crucial for the iron starvation state and was found to contain a degron, rescued the severe growth defect. Noteworthy, the - Fe state of HapX induced several gene clusters encoding secondary metabolites.

INTRODUCTION

Iron is an indispensable trace element which is required by all eukaryotic and virtually all prokaryotic species 1. The ability of iron to switch between two oxidation states, ferrous iron (Fe2+) and ferric iron (Fe3+), has made it an essential cofactor in redox biochemistry as heme, siroheme, iron-sulfur clusters, and mono- or dinuclear iron centers 2–5. Iron-dependent pathways include respiration, the TCA cycle, oxidative stress detoxification, P450 enzymes, DNA repair and replication as well as the biosynthesis of amino acids, nucleotides and sterols. Moreover, iron is also critically involved in less conserved pathways such as secondary metabolism 4. Although iron is essential, excess iron leads to the formation of highly reactive hydroxyl radicals through Haber-Weiss-Fenton chemistry causing oxidative cell damage 6,7. Therefore, sophisticated mechanisms are required to balance iron uptake, consumption, and storage to avoid both deficiency and toxicity of this metal, i.e. organisms must be able to discriminate cellular iron limitation (-Fe), iron sufficiency (+Fe) and iron excess (hFe) for respective adaptation.

Aspergillus fumigatus is one of the most common opportunistic fungal pathogens causing life-threatening invasive infectious diseases, particularly in immunocompromised patients, known as aspergillosis 8,9. Iron homeostasis has been shown to be key to the virulence of this mold in various infection models 4. In A. fumigatus iron homeostasis is mainly regulated by two iron-responsive transcription factors, termed HapX and SreA, which are interconnected in a negative transcriptional feedback loop 4. In -Fe, HapX represses iron-consuming pathways, including heme biosynthesis, respiration and vacuolar iron detoxification, while inducing high-affinity iron uptake through siderophore-mediated and reductive iron assimilation 10–12. hFe turns HapX into an activator of iron-consuming pathways and vacuolar iron deposition by the transporter CccA 13,14. During sufficient iron supply, SreA represses expression of HapX and high-affinity iron acquisition by siderophore-mediated and reductive iron assimilation 15,16. Consequently, loss of HapX impairs growth in -Fe and hFe but not +Fe conditions, whereas loss of SreA impairs growth in +Fe and especially in hFe but not in -Fe conditions 10,14,15,17.

SreA, a GATA-type zinc finger transcription factor, contains a single cysteine-rich region (CRR) for iron sensing 15,18–20. HapX shows several, evolutionary highly conserved characteristics: (i) a Hap4-like domain mediating direct interaction with the CCAAT-binding complex for cooperative DNA binding 12,21,22, (ii) the bZIP-type DNA-binding domain, (iii) a coiled-coil domain for dimerization, (iv) four CRRs, termed CRR-A to CRR-D that are involved in iron sensing, and (v) the C-terminal 27 amino acid region required for the role of HapX in -Fe but not hFe 4,14,21. Each CRR of SreA and HapX contains four cysteine residues, which differ in spacing and amino acid composition but show high phylogenetic conservation 4,23. Several lines of evidence indicate that regulation of iron homeostasis via SreA and HapX depends on [2Fe-2S] cluster sensing by the CRRs: (i) inactivation of mitochondrial [2Fe-2S], but not cytosolic [4Fe-4S] cluster biogenesis, impaired iron sensing in A. fumigatus 24; (ii) mutational analysis revealed that mainly CRR-B, but to a lesser extent also CRR-A and even less CRR-C, are required for HapX’s role in hFe but not -Fe, while mutation of CRR-D was phenotypically inconspicuous 14; (iii) GrxD, which belongs to the [2Fe-2S] cluster-transferring monothiol glutaredoxins 25, physically interacts with both HapX and SreA and appears to mediate the removal of [2Fe-2S] clusters from both HapX and SreA to switch HapX from the “iron” to the “-Fe” state and to inactivate SreA 17; (iv) the C-terminal domain of HapX (amino acids 161–491) containing the four CRRs, recombinantly expressed in Escherichia coli, exhibits a UV-vis spectrum indicative of [2Fe-2S] clusters 17; and (v) peptide mimics of the CRRs of HapX and SreA have the ability to coordinate [2Fe-2S] clusters in vitro with differing propensities, whereby the [2Fe-2S] cluster coordinated by HapX CRR-B showed an outstanding high stability 23. HapX and SreA are highly conserved in the fungal kingdom but not in Saccharomycotina, which employ different iron-sensing transcription factors 17. Remarkably, the GrxD homologous monothiol glutaredoxins Grx3 and Grx4 are required for adaptation to iron sufficiency in Saccharomyces cerevisiae in contrast to the situation in A. fumigatus, where GrxD is important for -Fe adaptation 17,26,27. These data highlight significant differences between iron homeostatic systems, even within fungal species.

Genetic inactivation of HapX causes growth and metabolic defects under -Fe and hFe but not +Fe 10,14, indicating that this regulator can switch between three distinct states, mediating adaptation to -Fe, +Fe (neutral/inactive) and hFe. Similar to siderophore biosynthesis and uptake 28–31, HapX is crucial for A. fumigatus virulence in a murine model of aspergillosis, highlighting -Fe in the host niche and the importance of pathogen adaptation 10. The latter demonstrates that switching HapX into its -Fe state is important for adaptation to the mammalian host niche. However, the molecular determinants of the HapX -Fe state have remained elusive. In this study, we elucidated the molecular mechanism for the HapX transitions between its three distinct functional states for adaptation to -Fe, +Fe (neutral/inactive), and hFe.

RESULTS

Combinatorial mutation of HapX CRR-A, CRR-B, CRR-C and CRR-D blocks growth

Previously, mutational analysis in HapX revealed that mainly CRR-B, but to a lesser extent also CRR-A and even less CRR-C, are required for HapX’s role in hFe but not -Fe, while mutation of CRR-D was phenotypically inconspicuous 14. Important to note, individual replacement of different cysteine residues of the same CRR by alanine had the same phenotypical consequences, indicating that the four cysteine residues in a CRR function in concert and that replacement of a single cysteine residue is sufficient to inactivate the function of a CRR 14. Attempts to generate a HapX allele expressed under its native promoter and lacking all four CRRs were unsuccessful, suggesting that this might be lethal. For further functional analysis of HapX protein domains, generated hapX alleles were therefore expressed under control of the xylose-inducible xylP promoter (PxylP), which allows tunable expression levels, including silencing and overexpression 32,33, with single copy integration at the native hapX locus. For phenotyping, fungal strains were grown on solid media reflecting -Fe, +Fe and hFe without xylose for repression and 0.1% xylose supplementation for expression of PxylP-driven hapX alleles (Figure 1). Compared to +Fe, A. fumigatus wildtype (wt) displayed reduced conidiation (reduced green coloration of the fungal colony) in -Fe and reduced radial growth in hFe, largely independent of xylose supplementation. Compared to wt, lack of HapX (ΔhapX) decreased conidiation in -Fe and blocked growth in hFe but showed wt-like growth in +Fe, independent of xylose supplementation, as previously reported 14. Without xylose supplementation, PxylP-driven native hapX displayed a ΔhapX growth phenotype, apart from slight growth in hFe most likely due to slight leakiness of PxylP 33. With xylose induction, PxylP-driven native hapX displayed a largely wt-like growth pattern (apart from mildly decreased growth in hFe), demonstrating the suitability of PxylP for studying functionality of hapX alleles.

Figure 1:
Figure 1: Combinatorial mutation of CRR-B and -C (strains hapXA/B/C/D, hapXA/B/C, hapXB/C/D, hapXB/C), but not their individual mutation (hapXB, hapXC), blocks growth independent of iron availability (-Fe / +Fe / hFe).

For every fungal strain, 104 conidia were spot-inoculated on minimal medium plates reflecting iron starvation (-Fe; 200 µM BPS), iron sufficiency (+Fe; 30 µM FeSO4) or excess iron (hFe; 10 mM FeSO4). Expression of PxylP-driven hapX alleles was tuned by supplementation with xylose: 0% for repression and 0.1% for moderate expression. Plates were incubated at 37 °C for 48 h. The CRRs and the C-terminus of the respective HapX alleles are schematically shown on the ride side; functional inactivation of a CRRs is indicated by white shading.

As a first step in the functional analysis of the HapX CRRs, we simultaneously replaced one cysteine of each CRR by an alanine residue (C203A, C277A, C353A, C380A) to block [2Fe-2S] cluster binding by all four CRRs, resulting in strain hapXA/B/C/D. Under non-inducing conditions (0% xylose), this strain showed a growth pattern similar to that of ΔhapX (Figure 1). Since the latter growth behavior was observed for all generated mutant strains without xylose supplementation (Figure 1), it is not discussed further for the other generated strains. Remarkably, xylose-induced expression of the hapXA/B/C/D allele blocked growth under all conditions tested (Figure 1). Importantly, individual mutation of the four CRRs of HapX expressed under the endogenous promoter was previously shown to negatively affect growth only in hFe, whereby this was observed mainly for CRR-B, less for CRR-A, only mildly for CRR-C and not at all for CRR-D 14. Similarly, under PxylP control, individual mutation of CRR-B (strain hapXB) blocked and that of CRR-C (strain hapXC) mildly decreased growth exclusively in hFe compared to the native hapX allele with xylose induction (Figure 1).

To investigate the reason for the growth blocking effect of hapXA/B/C/Dallele expression, we compared the expression of HapX target genes in strains expressing the PxylP-driven native hapX and hapXA/B/C/D alleles by Northern blot analysis. Therefore, hapX and hapXA/B/C/D strains were grown in +Fe liquid cultures without xylose supplementation for 17 h to circumvent the growth inhibitory effect of combinatorial mutation of all four CRRs, taking advantage of the phenotypically inconspicuous ΔhapX phenotype in +Fe (Figure 1), followed by incubation for 1 h in the presence of varying amounts of xylose to induce expression of the hapX alleles. Additionally, the wt was grown for 18 h in -Fe and +Fe liquid cultures as control. Harvested mycelia were subjected to Northern blot analysis of iron-regulated genes. In line with previously reported data 10,14, hapX transcript levels were strongly upregulated in -Fe compared to +Fe in wt (Figure 2A). In contrast, strains with PxylP-driven hapX showed hapX expression that increased with xylose supplementation from 0.05 to 1.0% xylose, whereas hapX transcripts were not detected without (0%) or low (0.01%) xylose supplementation (Figure 2B). In contrast to the hapX strain, with xylose induction ≥0.05%, the hapXA/B/C/D strain showed an expression pattern similar to that of the wt grown in -Fe (Figure 2B), i.e. induction of -Fe-induced mirB, which mediates siderophore uptake 34 and repression of -Fe-repressed heme biosynthetic hemA and iron regulatory sreA (Figure 2B). This expression pattern of strain hapXA/B/C/D is consistent with the previously reported -Fe response in A. fumigatus 10,14,15,17. These data strongly suggest that the simultaneous mutational disruption of all four HapX CRRs renders HapX iron-blind, regardless of the prevailing iron availability, as shown here for +Fe. As shown previously 15,18, sreA expression resulted in two different sized transcripts due to alternative transcription initiation. Notably, expression of hapXA/B/C/D mainly affected the larger sreA transcript (Figure 2B), revealing differential transcriptional regulation of the two sreA transcripts by HapX. Remarkably, high expression (1.0% xylose) of the wt hapX allele resulted in weak induction of mirB (Figure 2B).

Figure 2:
Figure 2: Combinatorial mutation of all four HapX CRRs (hapXA/B/C/D) results in an iron starvation response in +Fe.

Total RNA was isolated from liquid cultures grown either in +Fe or -Fe at 37 °C. The wt was grown for 18 h (A), strains expressing PxylP-driven hapX and hapXA/B/C/Dwere grown in +Fe without xylose for 17 h followed by 1 h incubation with different xylose concentrations for induction of hapX expression (B). Northern blot analyzed genes: hapX, iron limitation-induced mirB as well as iron limitation-repressed sreA and hemA. Ethidium bromide-stained rRNA is shown for loading and quality contol.

Combinatorial mutation of the CRRs locks HapX in the -Fe status causing cellular stress and affecting secondary metabolism

The Northern blot analysis of selected genes shown above (Figure 2) cumulatively suggested that combinatorial mutation of the four CRRs locks HapX in the -Fe state. To characterize the genome-wide effect of combinatorial mutation of the four CRRs, we employed high-throughput RNA sequencing for transcriptome profiling. Therefore, biological triplicates of strains encoding PxylP-driven hapX and the hapXA/B/C/D were grown in liquid culture in +Fe for 17 h to allow growth under non-inducing conditions followed by an incubation for 1 h in the presence of 0.1% xylose for induction of the hapX alleles. On average, we obtained 23,476,866 reads per individual sample. Genome alignment revealed an average unique mapping percentage of 82.5% to the A. fumigatus Af293 reference genome, identifying the expression of 9,859 genes, of which 1,406 were found to be upregulated and 1,175 were downregulated ≥ 2-fold in strain hapXA/B/C/D compared to hapX with an adjusted p-value < 0.05 (Figure 3). The transcriptome data are shown in Supplementary Table S1A; principal component and volcano plot analyses of the transcriptomic comparison are shown in Supplementary Figure S1.

Figure 3:
Figure 3: Heatmap comparison of selected differentially expressed genes in hapXA/B/C/D compared to hapX in +Fe.

Genes that have previously been shown to be directly downregulated by HapX in -Fe (A); genes involved in siderophore metabolism or localized in siderophore metabolic gene clusters that have previously been shown to be upregulated in -Fe (B); genes involved in stress response (C); genes involved in autophagy (D); and genes involved in secondary metabolism (E). Only genes downregulated with a log2 fold change ≤ -1 or upregulated with ≥ 1 are shown, with the exception of (E): here, all genes of respective secondary metabolite encoding gene clusters are shown. Fumarylalanine is synthesized by SidE, most likely without the need for other enzymes. Gene clusters are highlighted by grey shadded boxes on the left side of the heat maps. The respective transcriptome data subsets are shown in Supplementary Table S1B.

A previous study identified 33 genes that are directly repressed by the CBC:HapX complex in - Fe 12. These genes encode iron-linked steps in various pathways including respiration, TCA cycle, CccA-mediated vacuolar iron deposition, defense against oxidative and nitrosative stress by catalase and flavohemoglobin, SreA, as well as biosynthesis of heme, iron-sulfur clusters and amino acids. All of these genes were downregulated in hapXA/B/C/Dcompared to hapX (Figure 3A). Moreover, the majority of genes involved in siderophore-mediated iron assimilation or localized in encoding gene clusters, that have been previously shown to be repressed by SreA 15,17, were upregulated in hapXA/B/C/Dcompared to hapX (Figure 3B). Taken together, these results demonstrate that combinatorial mutation of the four CRRs locks HapX in the -Fe state leading to a transcriptional -Fe response in +Fe conditions. Therefore, the most likely reason for the severe effect of combinatorial mutation of the four CRRs described above is caused by uncontrolled iron acquisition combined with repression of iron-consuming pathways and CccA-mediated vacuolar iron detoxification.

Iron overload is expected to cause cellular stress, particularly oxidative and nitrosative stress due to the formation of highly reactive oxygen and nitrogen species, also known as Haber-Weiss-Fenton and Fenton-like chemistry 6,7,35,36. In line, several genes encoding stress-responsive regulatory or structural proteins were found to be upregulated in strain hapXA/B/C/D (Figure 3C), including the mitogen-activated protein kinase MpkB 37, the basic leucine zipper (bZIP) transcription factors AtfB and AtfD 38, glutathione biosynthetic GshA 24, several glutathione transferases (Gto1, GstA, GstB; 39, Cu/Zn-superoxide dismutase SodA 40 and nitrosative stress-detoxifying S-nitrosoglutathione reductase GnoA 41. Noteworthy, the genes encoding oxidative stress-detoxifying mycelial catalases Cat1 and Cat2 42 as well as nitrosative stress-detoxifying flavohemoglobin FhpA 41 were significantly downregulated in strain hapXA/B/C/D (Figure 3A). However, these genes have previously been shown to be directly repressed by HapX during -Fe 12,14. Consequently, the activation of the -Fe state of HapX in +Fe appears to result not only in uncontrolled iron uptake combined with repression of iron consumption and detoxification but most likely also impairs defense against oxidative and nitrosative stress.

Remarkably, the majority of the downregulated genes in strain hapXA/B/C/D are related to ribosome biogenesis and translation (Supplementary Figure S2 and Supplementary Table S1C), while five genes involved in autophagy were upregulated (Figure 3D), a pattern consistent with repression of TOR complex 1 (TORC1; target of rapamycin) 43,44.

In addition, biosynthetic gene clusters for the secondary metabolites fumigermin 45, pyomelanin/tyrosine degradation 46, fumarylalanine 47, helvolic acid 48, the meroterpenoid pyripyropene 49, fumitremorgins 50, and fumagillin/pseurotin 51,52 were upregulated in strain hapXA/B/C/D.

Notably, induction of fumarylalanine and pyomelanin/tyrosine degradation has previously been linked to cellular stress 46,47. In contrast, expression of gene clusters 53 for biosynthesis of fumigaclavines (fga), hexadehydroastechrome (has), isocyanides fumivaline/fumicicolin (crm), endocrocin (enc), trypacidin (tpc), xanthozillin (xan), fumisoquin (fmp/fsq), gliotoxin (gli), neosartoricin/fumicycline (nsc), trypacidin (tpc), fumihopaside (afum), satorypyrone (spy) was largely unaffected in strain hapXA/B/C/D compared to hapX (Supplementary Table S1A).

Combinatorial mutation of HapX CRR-B and CRR-C blocks growth and activates the -Fe state

To elucidate the combinatorial mutation of which of the four CRRs is responsible for the growth inhibitory effect, i.e. which CRRs are involved in locking HapX in the -Fe state, we generated mutant strains expressing hapX alleles carrying all combinatorial mutations of three CRRs, termed hapXA/B/C, hapXB/C/D, hapXA/B/D, and hapXA/C/D. Phenotyping revealed that only hapXA/B/C and hapXB/C/Dstrains phenocopied the hapXA/B/C/D strain upon xylose induction (Figure 1), suggesting that the combinatorial mutation of CRR-B and -C, which is common to these two alleles, is most likely responsible for the growth defect. Indeed, combinatorial mutation of only CRR-B and -C (strain hapXB/C) blocked growth upon xylose induction, regardless of the prevailing iron availability (Figure 1). In contrast, individual mutation of CRR-B (strain hapXB) or CRR-C (strain hapXC) or combinatorial mutation of CRR-A and -B (strain hapXA/B) impaired growth only in hFe (Figure 1). Taken together, these results demonstrate that combinatorial mutation of CRR-B and -C is sufficient to block growth, regardless of the prevailing iron availability. As previously shown in strains expressing hapX under its native promoter 14, individual mutation of CRR-B (strains hapXB, hapXA/B, hapXA/B/D) blocked and mutation of CRR-C (strain hapXC) reduced growth exclusively in hFe (Figure 1). Moreover, combinatorial mutation of CRR-A, -C and -D (strain hapXA/C/D) blocked growth in hFe (Figure 1). These results highlight the importance of CRR-A, -B and -C in mediating iron resistance in A. fumigatus, as previously demonstrated with individual mutation of CRRs 14. Mutation of CRR-D was phenotypically inconspicuous in all combinations tested (Figure 1).

All generated strains expressing hapX alleles were subjected to Northern blot analysis in +Fe with xylose induction for 1 h (Figure 4). In contrast to ΔhapX, all strains expressing PxylP-driven hapX alleles showed largely similar hapX induction (Figure 4A), whereby hapX transcript levels were slightly higher in strains hapXA/B/C/D, hapXA/B/C, hapXB/C/D compared to the other strains. Similar to the strain expressing hapX with combinatorial mutation of CRR-A, -B, -C and -D (hapXA/B/C/D), all other strains with combinatorial mutation of CRR-B and -C (strains hapXA/B/C, hapXB/C/Dand hapXB/C), in contrast to the remaining strains (ΔhapX, hapX, hapXA/B/D, hapXA/C/D, hapXA/B, hapXC, and hapXB), showed a transcriptional -Fe response in +Fe, i.e. higher transcript levels of mirB and lower transcript levels of hemA and the larger sreA transcript (Figure 4A), similar to wt in -Fe (Figure 4B). Again, mutation of CRR-D did not affect the transcriptional pattern in any combination. Taken together, these data underscore that the combinatorial mutation of CRR-B and -C blocks growth by causing "iron blindness" of HapX. In turn, these data suggest that CRR-B and -C do not coordinate [2Fe-2S] clusters in -Fe and that [2Fe-2S] cluster occupancy of CRR-B and/or -C switches HapX out of the iron starvation state.

Figure 4:
Figure 4: Combinatorial mutation of CRR-B and CRR-C (strains hapXA/B/C/D, hapXA/B/C, hapXB/C/D, hapXB/C) locks HapX in the iron limitation state, i.e. causes a transcriptional iron starvation response in +Fe.

Total RNA was isolated from liquid cultures grown in +Fe (A) or +Fe and -Fe (B) at 37 °C. Strains were grown in +Fe without xylose for 17 h followed by 1 h incubation with 0.1% xylose for induction of hapX expression (A) and wt was grown for 18 h without xylose supplementation (B). Northern blot analyzed genes: hapX, iron limitation-induced mirB as well as iron limitation-repressed sreA and hemA. Ethidium bromide-stained rRNA is shown for loading and quality contol.

C-terminal HapX truncation partially rescues the growth defect caused by combinatorial mutation of the four CRRs

The C-terminal 27 amino acid residues of HapX (amino acid residues 465-491) have previously been shown to be crucial adaptation to -Fe but not hFe 14. To investigate a possible link between the HapX C-terminus and the CRRs, we generated mutants expressing PxylP-driven hapX lacking the C-terminal 27 amino acid residues without (strain hapX464) or with combinatorial mutation of the four CRRs (hapXA/B/C/D;464). As previously shown with native promoter control 14, the C-terminal HapX truncation (strain hapX464) had no significant effect on growth in hFe (Figure 1). Remarkably, the C-terminal truncation partially rescued the growth defect caused by mutation of the four CRRs with xylose induction in -Fe and +Fe but not in hFe (Figure 1).

Northern blot analysis of mycelia grown in +Fe with xylose induction for 1 h revealed that truncation of the C-terminal 27 amino acid region (strain hapXA/B/C/D;464) attenuated the transcriptional iron starvation response caused by combinatorial mutation of the four CRRs (strain hapXA/B/C/D), i.e. it decreased the mirB transcript level and increased the level of the larger sreA transcript, while the hemA transcript level was not affected (Figure 5).

Figure 5:
Figure 5: C-terminal HapX truncation (strain hapXA/B/C/D,464) diminishes the transcriptional iron starvation response in +Fe caused by combinatorial mutation of the four CRRs (strain hapXA/B/C/D).

Total RNA was isolated from liquid cultures grown in +Fe at 37 °C for 17 h without xylose followed by 1 h incubation with 0.1% xylose for induction of PxylP-driven hapX alleles. Northern blot analyzed genes: hapX, iron limitation-induced mirB as well as iron limitation-repressed sreA and hemA. Ethidium bromide-stained rRNA is shown for loading and quality contol.

Taken together, the fact that the truncation of the C-terminal 27 amino acid residues, which have previously been shown to be crucial for -Fe adaptation 14, partially rescued the growth defect and attenuated the transcriptional -Fe response caused by combinatorial mutation of the four CRRs underlines that the growth defect is caused by the severe -Fe response.

Expression of hapX alleles with combinatorial mutation of CRR-B and -C mutation increases cellular iron accumulation

The transcriptomic analysis indicated that combinatorial mutation of CRR-B and -C causes upregulation of genes involved in iron uptake (Figures 2, 3, 4). To investigate intracellular iron accumulation, the mutant strains expressing the different hapX alleles were grown in +Fe liquid culture for 16 h without xylose induction, followed by another 6 h in the presence of xylose to induce expression of the different PxylP-driven hapX alleles. The schematic experimental setup is shown in Figure 6A. Optical inspection confirmed comparable biomass formation after 16 h of growth without xylose for all strains, consistent with the growth behavior on solid media (Figure 1). However, after the additional 6 h xylose induction period, all strains with combinatorial mutation of CRR-B and -C (hapXA/B/C/D, hapXA/B/C, hapXB/C/D and hapXB/C; hapXA/B/C/D;464), as well as hapXA/C/D and hapXC strains, showed significantly reduced biomass formation compared to hapXA/B/D, hapXA/B, hapXB and hapX464 and hapX (Figure 6B). These results further highlight the importance of HapX CRR-B and CRR-C for the fitness of A. fumigatus as seen in the plate growth assays (Figure 1). Among the strains with combinatorial mutation of CRR-B and -C, strain hapXA/B/C/D showed the most severe growth defect followed by hapXB/C, hapXA/B/C and hapXB/C/D. The statistically significant difference between hapXA/B/C/D and hapXA/B/C (Figure 6B) indicates a function of CRR-D, which was not seen in the growth analyses described above (Figure 1). The hapXA/B/C/D;464 strain displayed significantly increased biomass formation compared to the hapXA/B/C/D strain (Figure 6B), which is in agreement with the rescue of the growth inhibitory effect seen in the plate growth assays (Figure 1).

Figure 6:
Figure 6: Combinatorial mutation of CRR-B and -C (strains hapXA/B/C/D, hapXA/B/C, hapXB/C/D, hapXB/C) reduces biomass formation in liquid culture upon xylose supplementation and increases both cellular ferricrocin-complexed iron and the chelatable iron pool.

The schematic experimental setup is shown in (A). Liquid cultures were inoculated with the respective fungal strains and grown for 16 h at 37 °C in +Fe conditions, followed by supplementation with 1.0% xylose for PxylP induction and continued incubation for additional 6 h. After 22 h of incubation, the harvested biomass was used for estimation of biomass formation (B) and subsequently for the extraction of ferricrocin-complexed iron (C) and the determination of the chelatable iron pool (D). Values shown represent the mean ± standard deviation of biological triplicates. Significant differences, calculated by a two-way analysis of variance (ANOVA), are indicated by “x” for the reference strain hapX and “z” for the reference strain PxylP:hapXA/B/C/Dwith p < 0.0001.

Further analysis revealed that combinatorial mutation of CRR-B and -C increases cellular levels of iron complexed by the intracellular siderophore ferricrocin, a previously shown indicator of cellular iron accumulation 15,54,55: ∼ 7-fold in hapXA/B/C/D, ∼ 6-fold in hapXA/B/C and ∼ 2-fold in hapXB/C/D and hapXB/C compared to the hapX strain (Figure 6C). The statistically significant difference between hapXA/B/C/Dand hapXA/B/C again indicates a function of CRR-D. C-terminal truncation of HapX (strain hapXA/B/C/D;464) significantly reduced the accumulation of ferricrocin-complexed iron caused by combinatorial mutation of the four CRRs (strain hapXA/B/C/D). The individual mutation of CRR-C (hapXC), combinatorial mutation of CRR-A and -B (hapXA/B) and C-terminal truncation (hapX464) also resulted in slightly increased intracellular accumulation of ferricrocin-complexed iron (Figure 6C).

Another indicator of cellular iron overload is the chelatable iron pool, i.e. cellular iron not complexed by siderophores, which can be measured photometrically by reddish coloration due to chelation by a chelator such as BPS after reduction by ascorbic acid to ferrous iron 24. Only strains expressing hapX alleles with combinatorial mutation of CRR-B and -C showed significantly increased chelatable iron pools with hapXA/B/C/D> hapXA/B/C > hapXB/C/D = hapXB/C(Figure 6D). Again, the statistically significant difference between hapXA/B/C/D and hapXA/B/C indicates a function of CRR-D and C-terminal truncation (strain hapXA/B/C/D;464) significantly decreased the chelatable iron pool caused by combinatorial mutation of the four CRRs (strain hapXA/B/C/D). Remarkably, individual mutation of CRR-B reduced the chelatable cellular iron pool (Figure 6D).

Taken together, the increased levels in ferricrocin-complexed iron and chelatable iron in strains expressing hapX alleles with combinatorial mutation of CRR-B and -C are consistent with increased high-affinity iron uptake, as suggested by the gene expression analyses (Figures 2, 3, 4).

The C-terminus of HapX contains a degron

To analyze the consequences of the described mutations and C-terminal truncation on HapX protein abundance, we performed Western blot analysis in +Fe without xylose induction to allow growth of all mutant strains followed by a 1 h incubation in the presence of 0.1% xylose to induce expression of PxylP-driven hapX alleles (Figure 7). As reported previously 10,21, HapX was barely detectable in wt under +Fe conditions and was absent in the ΔhapX strain, while PxylP control increased the HapX protein level (Figure 7). Compared to the wt-like HapX (HapX), the different CRR mutations resulted in similar or slightly higher HapX protein levels, with the combinatorial mutation of CRR-A, -C and D (HapXA/C/D) resulting in the highest increase, approximately 6-fold, indicating increased stability of this HapX version as expression of all versions was driven by the same promoter (Figure 7). Remarkably, the truncation of the C-terminal 27 amino acid region, with (HapXA/B/C/D;464) or without (HapX464) combinatorial mutation of all four CRRs, increased the HapX protein levels approximately 10-fold (Figure 7), suggesting that the truncated region may contain a degron, a protein domain regulating the proteińs half-life by mediating its degradation 56. In agreement, fusion of the C-terminal 27 amino acid region of HapX to PxylP-driven Venus (strain VenusC-termHapX) significantly reduced Venus protein levels (Venus Mr = 26.8 kDa; VenusC-termHapX Mr = 30.3 kDa) in both -Fe and +Fe, as judged by both Venus-derived fluorescence of fungal colonies (Figure 8A) as well as the Venus protein levels in Western blot analysis (Figure 8B), but did not affect the transcript level (Figure 8C). Moreover, Western blot analysis clearly demonstrated degradation of the Venus-HapX fusion protein with the major degradation product having a molecular mass similar to that of Venus. Notably, Venus, like other green fluorescent protein relatives, forms an exceptionally stable 11-stranded β-barrel, which is difficult to degrade leading to quite stable intermediates that are still fluorescent 57. Therefore, the Venus protein might be a more stable degradation intermediate.

Figure 7:
Figure 7: Several mutations of CRRs and in particular C-terminal truncation increases the HapX protein level.

Strains were grown in +Fe without xylose for 17 h followed by 1 h incubation with 0.1% xylose for induction of hapX expression (same mycelia as used for Northern blot analysis shown in Figure 4) and subjected to Western blot analysis using an α-HapX antibody. All strains with PxylP-driven hapX alleles are shown in purple; HapX lacking ΔhapX in grey serves as the the negative control proving antibody specificity. Production of HapX from the native promoter (wt strain) is shown in black. Densitometrically estimated protein amounts were normalised to PxylP-driven wt-like HapX; numbers represent the mean ± standard deviation. Coomassie stained SDS-polyacrylamide gels are shown as a loading controls.

Figure 8:
Figure 8: The HapX C-terminus contains a degron.

C-terminal fusion of Venus with the C-terminal 27 amino acid region of HapX (strain VenusC-termHapX) decreases Venus-derived fluorescence intensity of fungal collonies (A) and protein stability in Western blot analysis (B), but does not affect transcript stability (C). Solid (A) and liquid (B) media contained 0.1% xylose for induction of PxylP-driven Venus alleles. On solid media, 104 conidia were spot-inoculated on medium reflecting iron starvation (-Fe; 200 µM BPS) or iron sufficiency (+Fe; 30 µM FeSO4) and incubated at 37 °C for 48 h (A). For Western blot analysis (B) using an α-GFP antibody (Venus Mr = 26.8 kDa; VenusC-termHapX Mr = 30.3 kDa), fungal strains were grown for 17 h in -Fe and +Fe liquid culture; -Fe did not contain BPS. Coomassie stained SDS-polyacrylamide gels are shown as a loading controls. Northern blot analysis was performed with a Venus-specific probe. Ethidium bromide-stained rRNA is shown for loading and quality contol.

DISCUSSION

HapX is a key regulator of iron homeostasis in A. fumigatus that is conserved in most filamentous ascomycetes. It is required for cellular adaptation to both -Fe and hFe, whereas it is dispensable for growth in +Fe. Thus, HapX appears to be able to switch between three functional states to mediate adaptation to -Fe, +Fe (neutral/inactive) and hFe. Its role during -Fe makes HapX important for the virulence of A. fumigatus in mammalian hosts and Fusarium oxysporum in plant hosts 10,58.

HapX contains four CRRs with four cysteine residues that differ in spacing and amino acid composition and are phylogenetically highly conserved 4,23. Previous studies have shown that iron regulation depends on mitochondrial [2Fe-2S] biosynthesis 24, that the C-terminal domain of HapX (amino acids 161–491) containing the four CRRs, recombinantly expressed in E. coli, exhibits a UV-vis spectrum indicative of the presence of [2Fe-2S] clusters 17, that the four CRRs of HapX can complex [2Fe-2S] clusters with different stabilities in vitro 23, and that the [2Fe-2S] cluster-transferring monothiol glutaredoxin GrxD physically interacts with HapX and appears to mediate removal of [2Fe-2S] clusters from HapX to switch it to the -Fe state 17. Taken together, these data suggest that HapX senses the cellular iron state by sensing the abundance of cellular [2Fe-2S] clusters. Consistent with this, mutation of mainly CRR-B, but to a lesser extent also CRR-A and even less CRR-C impaired hFe adaptation of A. fumigatus, while mutation of CRR-D had no effect 14. These results underline a role for three CRRs in the sensing of hFe by HapX. However, how HapX senses -Fe remained elusive, as individual mutation of any of the four CRRs did not affect -Fe adaptation of A. fumigatus 14.

Here, systematic combinatorial mutation of the HapX CRRs using conditional expression of the respective alleles with the xylose-inducible xylP promoter 32,33 revealed that expression of hapX alleles with combinatorial mutation of CRR-B and CRR-C (hapXA/B/C/D, hapXA/B/C, hapXB/C/D, hapXB/C), but not of other combinatorial CRR mutations (hapXA/B/D, hapXA/C/D, hapXA/B), nor individual mutation of CRR-B (hapXB) or CRR-C (hapXC), blocks growth regardless of iron availability, i.e. in -Fe, +Fe and hFe conditions (Figure 1). Transcriptome and individual gene expression analyses (Figures 2, 3, 4) revealed that combinatorial mutation of CRR-B and CRR-C results in an transcriptional -Fe response in +Fe, i.e. the induction of genes involved in siderophore-mediated iron acquisition 15,17 and the repression of all 33 previously identified HapX target genes involved in iron-consuming pathways 12. Taken together, these results suggest that combinatorial mutation of CRR-B and CRR-C locks HapX in the -Fe state. In other words, at least CRR-B and CRR-C, and possibly also CRR-A and -D, lack [2Fe-2S] in -Fe. Consequently, [2Fe-2S] cluster occupancy of CRR-B and/or -C switches HapX out of the -Fe state. Remarkably, of all four CRRs, CRR-B coordinates a [2Fe-2S] cluster with particularly high stability, by far higher than the other three CRRs 23. Therefore, CRR-B may be the first CRR sensing iron availability by binding a [2Fe-2S] cluster followed by CRR-C. As suggested by the growth defects in hFe caused by individual mutation of CRR-A, -B, and -C with endogenous promoter control 14 and with conditional expression (Figure 1), the switching of HapX to the hFe state then requires the complexation of a [2Fe-2S] clusters also by CRR-A. Notably, in all plate growth assays, mutation of CRR-D was phenotypically inconspicuous in all combinations tested.

In plate assays, all strains expressing hapX alleles with combinatorial mutation of at least CRR-B and -C blocked growth in all combinations (Figure 1). However, induction of these hapX alleles after uninduced pre-growth in liquid media revealed differences (Figure 6B): Strain hapXA/B/C/Dshowed the most severe growth defect followed by hapXB/C, hapXA/B/C and hapXB/C/D. The statistically significant difference between hapXA/B/C/Dand hapXA/B/C indicates a function of CRR-D, which was not seen in the growth analyses (Figure 1). Further analysis of these strains revealed that combinatorial mutation of CRR-B and -C increases cellular iron accumulation, i.e. it increased the cellular levels of iron complexed by the intracellular siderophore ferricrocin with hapXA/B/C/D > hapXA/B/C > hapXB/C/D > hapXB/C (Figure 6C) as well as the chelatable iron pool in a similar order with hapXA/B/C/D > hapXA/B/C > hapXB/C/D ≈ hapXB/C(Figure 6D). The statistically significant differences in both ferricrocin-complexed iron and the chelatable iron pool between hapXA/B/C/D and hapXA/B/Cunderline a function of CRR-D in iron sensing, which was not seen in the plate growth assays (Figure 1). Remarkably, individual mutation of CRR-B and, to a lesser extent, CRR-C reduced the chelatable cellular iron pool (Figure 6D). Both ferricrocin-complexed iron and the chelatable iron pool are previously shown indicators for iron overload 15,24,54,55. The increased cellular iron accumulation in strains expressing hapX alleles with combinatorial mutation of CRR-B and -C is consistent with the increased expression of genes involved in siderophore-mediated iron acquisition (Figures 2, 3, 4). Notably, all strains that showed an increased chelatable iron pool (Figure 6D) featured combinatorial mutation of CRR-B and -C and failed to grow on solid media regardless of iron availability (Figure 1), i.e. an increased iron pool was the best indicator of a growth defect in this experimental setup. This may be explained by the fact that the chelatable iron pool has a high potential to cause oxidative stress via Haber-Weiss/Fenton chemistry, whereas chelation of iron by siderophores is thought to prevent the Fenton reaction 7,59. Similar to strains expressing hapX alleles with combinatorial mutation of CRR-B and -C, depletion of the desulfurase Nfs1, which impairs mitochondrial [2Fe-2S] cluster biosynthesis and consequently iron sensing, was shown to result in a transcriptional -Fe response in combination with an increase in both ferricrocin-complexed iron and chelatable iron 24. These results underscore that combinatorial mutation of CRR-B and -C impairs iron sensing via [2Fe-2S] cluster perception.

Previously, the C-terminal 27 amino acid residues of HapX (amino acid residues 465-491) have been shown to be crucial for adaptation to -Fe but not to hFe 14, i.e. their truncation impaired growth, siderophore biosynthesis and repression of iron-consuming pathways in -Fe. Truncation of this C-terminal region (strain hapXA/B/C/D;464 compared to strain hapXA/B/C/D) partially rescued the growth defect caused by mutation of the four CRRs (Figure 1), attenuated the transcriptional -Fe response (Figure 5), and reduced both ferricrocin-complexed iron (Figure 6C) and the chelatable iron pool (Figure 6D). These results underline that the growth defect caused by combinatorial mutation of the four CRRs is indeed caused by the locked HapX -Fe state resulting in activation of high-affinity iron uptake combined with repressed iron consumption due to HapX-mediated repression of genes involved in iron consuming pathways, including repression of CccA-mediated iron detoxification by vacuolar iron deposition 13, leading to cellular iron overload and cellular stress. Consistent with these findings, combined mutation of all CRRs caused transcriptional upregulation of several genes encoding stress-responsive regulatory or structural proteins including the mitogen-activated protein kinase MpkB, the basic leucine zipper (bZIP) transcription factors AtfB and AtfD glutathione biosynthetic GshA, several glutathione transferases (Gto1, GstA, GstB), Cu/Zn-superoxide dismutase SodA and nitrosative stress-detoxifying S-nitrosoglutathione reductase GnoA (Figure 3C). Notably, the genes encoding oxidative stress-detoxifying mycelial catalases Cat1 and Cat2 as well as nitrosative stress-detoxifying flavohemoglobin FhpA were significantly downregulated (Figure 3A). However, these genes have previously been shown to be directly repressed by HapX during -Fe 12,14. Consequently, the activation of the -Fe state of HapX in +Fe also impairs defense against oxidative and nitrosative stress. The transcriptome analysis also revealed the downregulation of numerous genes involved in ribosome biogenesis and translation (Supplementary Figure S2 and Supplementary Table S1C), and upregulation of five genes involved in autophagy (Figure 3D). This pattern is consistent with repression of TOR complex 1 (TORC; target of rapamycin), a nutrient-sensitive, central regulator of cell growth. Starvation, stress and the drug rapamycin repress TORC1, resulting in repression of protein synthesis, induction of autophagy, exit from the cell cycle, and entry into a quiescent G0 state 43,44. Therefore, repression of TORC1 may also be involved in growth inhibition by combinatorial mutation of at least CRR-B and -C.

Western blot analysis revealed that truncation of the C-terminal 27 amino acid residues, with or without combinatorial mutation of all four CRRs, increased the HapX protein levels approximately 10-fold (Figure 7), suggesting that the truncated region may contain a degron, a protein domain regulating the protein’s half-life by mediating its degradation 56. In agreement, C-terminal tagging of Venus with the C-terminal 27 amino acid region of HapX significantly reduced Venus protein levels in both -Fe and +Fe, as judged by both Venus-derived fluorescence of fungal colonies (Figure 8A) and Venus protein levels (Figure 8B). Notably, Venus like other green fluorescent protein relatives forms an exceptionally stable 11-stranded β-barrel, which is difficult to degrade leading to quite stable intermediates that are still fluorescent 57. Previously, the HapX protein was shown to have a very short half-life of about 15 minutes in -Fe, to be post-translationally modified by ubiquitination, sumoylation and phosphorylation and to physically interact with the monothiol glutaredoxin GrxD and the F-box protein Fbx22 17,60. This newly identified degron represents a new factor influencing the protein stability of HapX and illustrates the complex post-translational regulation of this transcription factor.

Interestingly, the transcriptome analysis also indicated that combinatorial mutation of all CRRs causes upregulation of several gene clusters for biosynthesis of secondary metabolites, i.e. fumarylalanine, pyomelanin/tyrosine degradation, helvolic acid, fumigermin, meroterpenoid pyripyropene, fumitremorgins, and fumagillin/pseurotin (Figure 3E). These gene clusters may be directly activated by the -Fe state of HapX, by the general -Fe response, or by the induced stress that ultimately blocks growth. Notably, the induction of fumarylalanine and pyomelanin/tyrosine degradation has previously been linked to cellular stress 46,47.

Taken together, the present study suggests a model for the switching of HapX between its three functional states by the different propensities of the four CRRs for [2Fe-2S] cluster coordination dependent on iron availability. In a living cell, there are multiple HapX molecules that are unlikely to all be in the same functional state, allowing fine-tuning of regulation through the distribution of the three functional states. Most of the HapX homologs from filamentous ascomycetes comprise several CRRs, with CRR-B being the most highly conserved one, suggesting a similar mechanism to A. fumigatus HapX 14,23. However, there are transcription factors that share only some domains and thus not all functions with HapX: S. cerevisiae Yap5 has only two [2Fe-2S] cluster-coordinating CRRs, one of which is highly similar to HapX-CRR-B (67% identity at the amino acid level) and is required exclusively for hFe resistance. In contrast, Schizosaccharomyces pombe Php4, which is involved exclusively in adaptation to -Fe, lacks any classical CRR and contains two cysteine residues for binding of a bridging [2Fe–2S] cluster in cooperation with a monothiol glutaredoxin 4,61. This data illustrates the modular toolbox used in iron-regulatory transcription factors and underlines the requirement of multiple CRRs for transcription factors enabling adaptation to both -Fe and hFe conditions.

AUTHOR CONTRIBUTIONS

S.O.: conceptualization, methodology, investigation, data curation and evaluation, visualization, writing – original draft, writing – review and editing.

M.M.: conceptualization, methodology.

H.H.: conceptualization, funding acquisition, investigation, project administration, resources, supervision, visualization, writing – original draft, writing – review and editing.

DECLARATION OF AI USE

We have not used AI-assisted technologies in creating this manuscript.

CONFLICTS OF INTEREST

The authors declare no conflict of interests.

MATERIAL and METHODS

GROWTH CONDITIONS

Aspergillus minimal medium 62 containing 1% (w/v) glucose and 20 mM glutamine as carbon and nitrogen sources, respectively, supplemented with iron-free trace elements was used to grow A. fumigatus strains. For phenotyping under different iron availabilities on solid media, media were supplemented with 0.2 mM bathophenanthrolinedisulfonic acid (BPS) without iron addition to limit bioavailability and to inhibit reductive iron assimilation 63 for -Fe conditions, 0.03 mM FeSO4 to reflect +Fe, or 10 mM FeSO4 to mimic hFe. For solid media, 1.8% (w/v) agar was added, and 104 spores were point-inoculated. For liquid cultures, 100 mL minimal medium in 500 mL Erlenmeyer flasks was inoculated with 108 spores and shaken at 200 revolutions per minute. BPS was not added in -Fe liquid cultures. For induction of the conditional xylP promoter (PxylP), xylose was added to a final concentration of 0.1% (w/v) 32,33. Cultures were generally incubated at 37 °C.

MUTANT STRAIN GENERATION

In this study, the non-homologous end joining-lacking (ΔakuA::loxP) ATCC46645 derivative AfS77, here referred to as wt, was used 64. Nucleic acid fragments for generation of plasmids were purified either by column purification or by gel extraction (Monarch PCR & DNA Cleanup Kit and Monarch DNA Gel Extraction Kit, New England Biolabs). Fragments were assembled using the NEBuilder HiFi DNA Assembly or applying site-directed mutagenesis (New England Biolabs); resulting plasmids were amplified in E. coli 5-alpha and purified using the Monarch Plasmid Miniprep Kit (New England Biolabs).

For generation of a strain expressing PxylP-driven hapX (strain hapX), the 1 kb hapX 5’-non-coding region for hapX locus-specific targeting was amplified from wt genomic DNA using primers 5’hapX flank_fwd/5’hapX flank_rev, the hph-PxylP cassette was amplified from genomic DNA of strain hapXOE 60 using primers hph-xylP cassette_fwd/hph-xylP cassette_rev and the hapX gene (Afu5g03920) including its native 1 kb 3’-non-coding region was amplified from wt genomic DNA using primers hapX-3’flank_fwd/hapX-3’flank_rev. These fragments were assembled with the backbone of the commercially available plasmid pJet1.2 (Thermo Fisher Scientific) amplified using primers pJet1.2 backbone_fwd/pJet1.2 backbone_rev applying the NEBuilder approach, yielding plasmid pSO11. The hph-PxylP cassette contains the hygromycin resistance cassette 65, hph, and the xylose-inducible xylP promoter, termed PxylP 32, which allows conditional expression of the hapX alleles.

The plasmid for the hapX version with combinatorial mutations of all four CRRs was based on plasmid pSO11 and was generated by site-directed mutagenesis. Therefore, a single cysteine residue of each CRR was mutated to an alanine residues (C203A, C277A, C353A, C380A) using mutagenesis primers, i.e. oAfHapX-A2.f/oAfHapX-A2.r, oAfHapX-B1.f/oAfHapX-B1.r, oAfHapX-C3.f/oAfHapX-C3.r and oAfHapX-D2.f/oAfHapX-D2.r. The resulting plasmid pSO03, encoding the hapX allele designated hapXA/B/C/D(mutated CRRs are indicated in superscript letters), and plasmid pSO11 were subsequently used as templates to generate plasmids encoding the different combinations of CRR mutations; where appropriate, previous cysteine-to-alanine mutations were reversed to the required combinations of CRR mutations. The construction of the other plasmids is not described separately as they were constructed using the site directed mutagenesis strategy as described for pSO03. C-terminal truncations of hapX and hapXA/B/C/D, resulting in hapX464 and hapXA/B/C/D;464, were also generated using the site directed mutagenesis strategy using primers oAfhapX-SO7/oAfhapX-SO8. For fungal transformation the transformation cassettes were PCR-amplified from respective plasmids using primers oAfhapX-1/oAfhapX-2. After fungal transformation 66, transformants were selected using 100 µg/mL hygromycin. For the generation of strains for PxylP-driven expression of Venus and a fusion protein of Venus and the C-terminal part of HapX (VenusC-termHapX), plasmid pSO52 was generated. Therefore, the following fragments were PCR amplified and assembled using the NEBuilder HiFi DNA Assembly (New England Biolabs): (i) the plasmid backbone containing the fcyB 5’- and 3’-non-coding regions, the PxylP and the trpC terminator derived from plasmid pSO16 67 using primers fcyB-bb_trpC_fwd/fcyB-bb_xylP_rev; (ii) the Venus coding sequence derived from plasmid pSO16 67 with the addition of a short linker sequence using primers Venus_fwd/ Venus_linker-seq_rev; and (iii) the C-terminal hapX coding region from wt gDNA using primers linker-seq_hapX_fwd/hapX_rev. Plasmid, pSO52 was then used as a PCR template to generate plasmids pSO53 and pSO54. Plasmid pSO53, encoding Venus only, was generated by site-directed mutagenesis using primers pSO53_fwd/pSO53_rev, deleting the hapX coding sequence. Plasmid pSO54, encoding Venus fused with the C-terminal 27 amino acid region of HapX, was generated by site-directed mutagenesis using primers pSO54_fwd/pSO54_rev. For fungal transformation, the plasmids were linearized by NotI restriction digestion and purified by column purification. In this case, no additional selection marker was required as locus-specific integration at the fcyB locus was used 68. Therefore, transformants were selected using 10 µg/mL 5-fluorcytosine.

The generated strains, plasmids and primers used strains used in this study are summarized in Supplementary Table S2, S3 and S4, respectively. All generated mutant strains were confirmed by Southern blot analysis; the results of the Southern blot analyses and schematic illustrations of the generated genotypes are shown in Figure S3.

ISOLATION of RNA and DNA; SOUTHERN and Northern BLOT ANALYSIS

Genomic DNA was isolated by PCI extraction and isopropanol precipitation. For Southern blot analysis, DNA digested with specified restriction enzymes, was separated on a 0.7% (w/v) agarose gel and blotted on a HybondTM-N membrane (Cytiva AmershamTM) with NaOH for denaturation.

Total RNA was extracted from freshly harvested biomass using TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. For RNA gel electrophoresis, 10 µg of isolated RNA were loaded onto 1.2% (w/v) agarose gels with 1.85% (w/v) formaldehyde. For Northern blot analysis, HybondTM-N+ membranes (Cytiva AmershamTM) were used.

Hybridization probes for Southern and Northern blot analyses were generated by PCR amplification using digoxigenin-labelled deoxyribonucleotides (Roche); used primers are listed in Supplementary Table S4 and S5, respectively.

TRANSCRIPTOM ANALYSIS USING RNA SEQUENCING

Illumina RNA library preparation, miCORE mRNA sequencing and bioinformatic analysis was performed by Microsynth AG. Illumina reads were mapped to the reference genome, A. fumigatus Af293 ASM265 (NCBI RefSeq assembly: GCF_000002655.1) downloaded from NCBI database 69 using STAR version 2.7.11b 70. The gene counts were normalized applying DESeq2 71. Differentially expressed genes were filtered using log2 fold change ≤ -1 or ≥ 1 and adjusted p-value < 0.05 as cut-offs. Visualization of the differential gene expression patterns in heat maps was performed using GraphPad Prism version 10.2.3 (GraphPad Software, www.graphpad.com). For functional categorization of the differentially expressed genes, the web-based tool FungiFun (version 2.2.8 BETA) was used 72.

DETERMINATION OF THE CELLULAR FERRICROCIN CONTENT AND THE CHELATABLE IRON POOL

Cellular contents of iron-loaded ferricrocin were determined as described previously 24,73. 80 mg lyophilized mycelium was homogenized and resuspended in 1.8 mL sodium-phosphate buffer [pH 7.5; 50 mM] and incubated for 30 min on ice. Cellular debris was removed by centrifugation and 0.8 mL of the supernatant was transferred to fresh tubes and mixed with 0.2 mL of ROTI® phenol/chloroform/isoamyl alcohol (50:24:1; Roth). After phase separation by centrifugation, 0.1 mL of the organic phase was transferred to fresh tubes and mixed with 0.1 mL water and 0.5 mL diethyl ether. After vigorous mixing and centrifugation, the ferricrocin concentration of the lower aqueous phase was measured at a wavelength of 434 nm using a nano spectrophotometer (Thermo Fisher Scientific).

The chelatable iron pool of was quantified in the ferricrocin-depleted supernatant after ROTI® phenol/chloroform/isoamyl alcohol (50:24:1; Roth) extraction and addition of BPS and ascorbic acid to final concentrations of 1 mM each, as previously described 24. BPS-complexed ferrous iron was recorded at a wavelength of 535 nm using a molar extinction coefficient of e = 17,000 L mol-1 cm-1.

PROTEIN EXTRACTION and WESTERN BLOT ANALYSIS

Total protein was extracted from lyophilized biomass (Triad freeze dryer, Labconco) by alkaline lysis, in combination with trichloroacetic acid mediated protein precipitation, as reported previously 74. Total protein extracts were separated electrophoretically on 12 % (w/v) SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (AmershamTM ProtranTM Premium 0.45 µm NC, Cytiva AmershamTM) using the Trans-Blot® Turbo™ System (Bio-Rad Laboratories).

HapX proteins were recorded using a rabbit α-HapX antibody 17, 1:10,000 diluted, in combination with a secondary, peroxidase-coupled α-rabbit antibody (Sigma-Aldrich), 1:20,000 diluted, using ECL detection (Cytiva AmershamTM). Protein amounts were estimated densitometrically using Fiji ImageJ 75.

Venus proteins were detected using a mouse α-GFP antibody (1:10,000 diluted; Roche) as the primary antibody in combination with a peroxidase-coupled secondary anti-mouse antibody (1:10,000 diluted; Sigma-Aldrich). The ECL reagent (Cytiva AmershamTM) was used for detection.

BIOINFORMATICS

Required gene and protein sequences were downloaded from FungiDB 76 or from the NCBI database 69. Plasmid and protein sequences were generated and maintained using the cloud-based platform Benchling (Biology Software, 2023). Sequence alignments of genes and proteins were generated using Geneious Prime (2024.0.5; https://www.geneious.com) 77.

STATISTICAL ANALYSES

Statistical analyses were carried out applying GraphPad Prism version 10.2.3 (GraphPad, www.graphpad.com).

ACKNOWLEDGEMENTS and FUNDING

This research was funded by the Austrian Science Fund (FWF) [Grant-DOI: 10.55776/DOC82] to H.H. For open access purposes, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.

The authors acknowledge support from COST Action (European Cooperation in Science and Technology), FeSImmChemNet, CA21115.

ABBREVIATIONS

BPS
bathophenanthroline disulfonic acid
CRR
cysteine-rich region
-Fe
iron-limiting condition
+Fe
iron sufficient condition
hFe
iron excess condition
PxylP
xylose-inducible xylP promoter
wt
wild type;

REFERENCES

  1. 1.↵
    Kaplan, C. D. & Kaplan, J. Iron acquisition and transcriptional regulation. Chem Rev 109, 4536–4552 (2009).
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Mühlenhoff, U. et al. Compartmentalization of iron between mitochondria and the cytosol and its regulation. Eur J Cell Biol 94, 292–308 (2015).
    OpenUrlCrossRefPubMed
  3. 3.
    Dietl, A.-M., Binder, U., Shadkchan, Y., Osherov, N. & Haas, H. Siroheme is essential for assimilation of nitrate and sulfate as well as detoxification of nitric oxide but dispensable for murine virulence of Aspergillus fumigatus. Front Microbiol 9, (2018).
  4. 4.↵
    Misslinger, M., Hortschansky, P., Brakhage, A. A. & Haas, H. Fungal iron homeostasis with a focus on Aspergillus fumigatus. Biochim Biophys Acta Mol Cell Res 1868, 118885 (2021).
    OpenUrl
  5. 5.↵
    Philpott, C. C. et al. Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool. Front Mol Biosci 10, 1127690 (2023).
  6. 6.↵
    Haber, F., Weiss, J. & Pope, W. J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond A Math Phys Sci 147, 332–351 (1997).
    OpenUrl
  7. 7.↵
    Halliwell, B. & Gutteridge, J. M. Role of iron in oxygen radical reactions. Methods Enzymol 105, 47–56 (1984).
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    Brown, G. D. et al. Hidden killers: human fungal infections. Sci Transl Med 4, 165rv13 (2012).
    OpenUrlCrossRef
  9. 9.↵
    Arastehfar, A., et al. Aspergillus fumigatus and aspergillosis: from basics to clinics. Stud Mycol 100, 100115 (2021).
  10. 10.↵
    Schrettl, M. et al. HapX-mediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus. PLoS Pathog 6, e1001124 (2010).
    OpenUrlCrossRefPubMed
  11. 11.
    Blatzer, M., Binder, U. & Haas, H. The metalloreductase FreB is involved in adaptation of Aspergillus fumigatus to iron starvation. Fungal Genet Biol 48, 1027–1033 (2011).
    OpenUrlCrossRefPubMed
  12. 12.↵
    Furukawa, T. et al. The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition. Nucleic Acids Res 48, 3567–3590 (2020).
    OpenUrlCrossRefPubMed
  13. 13.↵
    Gsaller, F. et al. The interplay between vacuolar and siderophore-mediated iron storage in Aspergillus fumigatus. Metallomics 4, 1262 (2012).
    OpenUrlCrossRefPubMed
  14. 14.↵
    Gsaller, F. et al. The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess. EMBO J 33, 2261–2276 (2014).
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Schrettl, M. et al. SreA-mediated iron regulation in Aspergillus fumigatus. Mol Microbiol 70, 27–43 (2008).
    OpenUrlCrossRefPubMed
  16. 16.↵
    Yap, A., Volz, R., Paul, S., Moye-Rowley, W. S. & Haas, H. Regulation of high-affinity iron acquisition, including acquisition mediated by the iron permease FtrA, is coordinated by AtrR, SrbA, and SreA in Aspergillus fumigatus. mBio 14, e00757–23 (2023).
    OpenUrlPubMed
  17. 17.↵
    Misslinger, M. et al. The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus. PLoS Genet 15, e1008379 (2019).
    OpenUrlCrossRefPubMed
  18. 18.↵
    Haas, H., Zadra, I., Stöffler, G. & Angermayr, K. The Aspergillus nidulans GATA factor SREA is involved in regulation of siderophore biosynthesis and control of iron uptake. J Biol Chem 274, 4613–4619 (1999).
    OpenUrlAbstract/FREE Full Text
  19. 19.
    Harrison, K. A. & Marzluf, G. A. Characterization of DNA binding and the cysteine rich region of SRE, a GATA factor in Neurospora crassa involved in siderophore synthesis. Biochemistry 41, 15288–15295 (2002).
    OpenUrlCrossRefPubMed
  20. 20.↵
    Pelletier, B., Trott, A., Morano, K. A. & Labbé, S. Functional characterization of the iron-regulatory transcription factor Fep1 from Schizosaccharomyces pombe. J Biol Chem 280, 25146–25161 (2005).
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Hortschansky, P. et al. Interaction of HapX with the CCAAT-binding complex - a novel mechanism of gene regulation by iron. EMBO J 26, 3157–3168 (2007).
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Huber, E. M. et al. Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX. Structure 30, 934–946.e4 (2022).
    OpenUrlCrossRef
  23. 23.↵
    Rossetto, D. et al. Peptide mimics of the cysteine-rich regions of HapX and SreA bind a [2Fe-2S] cluster in vitro. Adv Biol 8, 2300545 (2024).
  24. 24.↵
    Misslinger, M., Lechner, B. E., Bacher, K. & Haas, H. Iron-sensing is governed by mitochondrial, not by cytosolic iron–sulfur cluster biogenesis in Aspergillus fumigatus. Metallomics 10, 1687–1700 (2018).
    OpenUrlCrossRefPubMed
  25. 25.↵
    Talib, E. A. & Outten, C. E. Iron-sulfur cluster biogenesis, trafficking, and signaling: roles for CGFS glutaredoxins and BolA proteins. Biochim Biophys Acta Mol Cell Res 1868, 118847 (2021).
    OpenUrl
  26. 26.↵
    Li, H. et al. The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation. Biochemistry 48, 9569–9581 (2009).
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    Li, H. & Outten, C. E. The conserved CDC motif in the yeast iron regulator Aft2 mediates iron–sulfur cluster exchange and protein–protein interactions with Grx3 and Bol2. J Biol Inorg Chem 24, 809–815 (2019).
    OpenUrlCrossRefPubMed
  28. 28.↵
    Chamilos, G. et al. Exploring the concordance of Aspergillus fumigatus pathogenicity in mice and Toll-deficient flies. Med Mycol 48, 506–510 (2010).
    OpenUrlCrossRefPubMed
  29. 29.
    Schrettl, M. et al. The crucial role of the Aspergillus fumigatus siderophore system in interaction with alveolar macrophages. Microbes Infect 12, 1035–1041 (2010).
    OpenUrlCrossRefPubMed
  30. 30.
    Slater, J. L., Gregson, L., Denning, D. W. & Warn, P. A. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med Mycol 49 Suppl 1, S107–113 (2011).
    OpenUrlCrossRefPubMed
  31. 31.↵
    Happacher, I., Aguiar, M., Yap, A., Decristoforo, C. & Haas, H. Fungal siderophore metabolism with a focus on Aspergillus fumigatus: impact on biotic interactions and potential translational applications. Essays Biochem EBC20220252 (2023) doi:10.1042/EBC20220252.
    OpenUrlCrossRef
  32. 32.↵
    Zadra, I., Abt, B., Parson, W. & Haas, H. xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE. Appl Environ Microbiol 66, 4810– 4816 (2000).
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Yap, A., Glarcher, I., Misslinger, M. & Haas, H. Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species. Metab Eng Commun 15, e00214 (2022).
    OpenUrl
  34. 34.↵
    Aguiar, M. et al. Uptake of the siderophore triacetylfusarinine C, but not fusarinine C, is crucial for virulence of Aspergillus fumigatus. mBio 13, e02192–22 (2022).
    OpenUrlPubMed
  35. 35.↵
    De Freitas, J. et al. Yeast, a model organism for iron and copper metabolism studies. Biometals 16, 185–197 (2003).
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    Rodrigues-Pousada, C. et al. Yeast AP-1 like transcription factors (Yap) and stress response: a current overview. Microb Cell 6, 267–285 (2019).
    OpenUrlCrossRefPubMed
  37. 37.↵
    Manfiolli, A. O. et al. Mitogen-activated protein kinase cross-talk interaction modulates the production of melanins in Aspergillus fumigatus. mBio 10, e00215–19 (2019).
    OpenUrlCrossRefPubMed
  38. 38.↵
    Silva, L. P., Horta, M. A. C. & Goldman, G. H. Genetic interactions between Aspergillus fumigatus basic leucine zipper (bZIP) transcription factors AtfA, AtfB, AtfC, and AtfD. Front Fungal Biol 2, 632048 (2021).
  39. 39.↵
    Burns, C. et al. Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus. Fungal Genet Biol 42, 319–327 (2005).
    OpenUrlCrossRefPubMed
  40. 40.↵
    Oberegger, H., Zadra, I., Schoeser, M. & Haas, H. Iron starvation leads to increased expression of Cu/Zn-superoxide dismutase in Aspergillus. FEBS Lett 485, 113–116 (2000).
    OpenUrlCrossRefPubMedWeb of Science
  41. 41.↵
    Lapp, K. et al. Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence. Front Microbiol 5, 469 (2014).
  42. 42.↵
    Paris, S. et al. Catalases of Aspergillus fumigatus. Infect Immun 71, 3551–3562 (2003).
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    Raught, B., Gingras, A. C. & Sonenberg, N. The target of rapamycin (TOR) proteins. Proc Natl Acad Sci U S A 98, 7037–7044 (2001).
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    Loewith, R. & Hall, M. N. Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189, 1177–1201 (2011).
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    Stroe, M. C. et al. Targeted induction of a silent fungal gene cluster encoding the bacteria-specific germination inhibitor fumigermin. Elife 9, e52541 (2020).
    OpenUrlCrossRefPubMed
  46. 46.↵
    Heinekamp, T., et al. Aspergillus fumigatus melanins: interference with the host endocytosis pathway and impact on virulence. Front Microbiol 3, 440 (2012).
  47. 47.↵
    Steinchen, W. et al. Bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus. Appl Environ Microbiol 79, 6670–6676 (2013).
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    Lv, J.-M. et al. Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis. Nat Commun 8, 1644 (2017).
    OpenUrlCrossRefPubMed
  49. 49.↵
    Itoh, T. et al. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases. Nat Chem 2, 858–864 (2010).
    OpenUrlCrossRefPubMed
  50. 50.↵
    Maiya, S., Grundmann, A., Li, S.-M. & Turner, G. The fumitremorgin gene cluster of Aspergillus fumigatus: identification of a gene encoding brevianamide F synthetase. Chembiochem 7, 1062–1069 (2006).
    OpenUrlCrossRefPubMedWeb of Science
  51. 51.↵
    Wiemann, P. et al. Prototype of an intertwined secondary-metabolite supercluster. Proc Natl Acad Sci U S A 110, 17065–17070 (2013).
    OpenUrlAbstract/FREE Full Text
  52. 52.↵
    Steenwyk, J. L. et al. Variation among biosynthetic gene clusters, secondary metabolite profiles, and cards of virulence across Aspergillus species. Genetics 216, 481–497 (2020).
    OpenUrlAbstract/FREE Full Text
  53. 53.↵
    Seo, H.-W. et al. A timeline of biosynthetic gene cluster discovery in Aspergillus fumigatus: from characterization to future perspectives. J Fungi (Basel) 10, 266 (2024).
  54. 54.↵
    Eisendle, M. et al. The intracellular siderophore ferricrocin is involved in iron storage, oxidative-stress resistance, germination, and sexual development in Aspergillus nidulans. Eukaryot Cell 5, 1596–1603 (2006).
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    Schrettl, M. et al. Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection. PLoS Pathog 3, 1195–1207 (2007).
    OpenUrlCrossRefPubMedWeb of Science
  56. 56.↵
    Ella, H., Reiss, Y. & Ravid, T. The hunt for degrons of the 26S proteasome. Biomolecules 9, 230 (2019).
  57. 57.↵
    Bragança, C. E. & Kraut, D. A. Mode of targeting to the proteasome determines GFP fate. J Biol Chem 295, 15892–15901 (2020).
    OpenUrlAbstract/FREE Full Text
  58. 58.↵
    López-Berges, M. S. et al. HapX-mediated iron homeostasis is essential for rhizosphere competence and virulence of the soilborne pathogen Fusarium oxysporum. Plant Cell 24, 3805–3822 (2012).
    OpenUrlAbstract/FREE Full Text
  59. 59.↵
    Gutteridge, J. M. & Halliwell, B. Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 899, 136–147 (2000).
    OpenUrlCrossRefPubMedWeb of Science
  60. 60.↵
    López-Berges, M. S. et al. The bZIP transcription factor HapX is post-translationally regulated to control iron homeostasis in Aspergillus fumigatus. Int J Mol Sci 22, 7739 (2021).
    OpenUrlCrossRefPubMed
  61. 61.↵
    Labbé, S., Khan, M. G. M. & Jacques, J.-F. Iron uptake and regulation in Schizosaccharomyces pombe. Curr Opin Microbiol 16, 669–676 (2013).
    OpenUrlCrossRefPubMed
  62. 62.↵
    Pontecorvo, G., Roper, J. A., Hemmons, L. M., Macdonald, K. D. & Bufton, A. W. J. The genetics of Aspergillus nidulans. Adv Genet 5, 141–238 (1953).
    OpenUrlCrossRefPubMedWeb of Science
  63. 63.↵
    Schrettl, M. et al. Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J Exp Med 200, 1213–1219 (2004).
    OpenUrlAbstract/FREE Full Text
  64. 64.↵
    Hartmann, T. et al. Validation of a self-excising marker in the human pathogen Aspergillus fumigatus by employing the β-rec/six site-specific recombination system. Appl Environ Microbiol 76, 6313–6317 (2010).
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    Punt, P. J., Oliver, R. P., Dingemanse, M. A., Pouwels, P. H. & van den Hondel, C. A. M. J. J. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56, 117–124 (1987).
    OpenUrlCrossRefPubMedWeb of Science
  66. 66.↵
    Tilburn, J. et al. Transformation by integration in Aspergillus nidulans. Gene 26, 205–221 (1983).
    OpenUrlCrossRefPubMedWeb of Science
  67. 67.↵
    Oberegger, S. et al. The cytosolic form of dual localized BolA family protein Bol3 is important for adaptation to iron starvation in Aspergillus fumigatus. Open Biol 14, 240033 (2024).
  68. 68.↵
    Birštonas, L. et al. Multiplex genetic engineering exploiting pyrimidine salvage pathway-based endogenous counterselectable markers. mBio 11, doi:10.1128/mbio.00230-20 (2020).
    OpenUrlCrossRef
  69. 69.↵
    Sayers, E. W. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 47, D23–D28 (2019).
    OpenUrlCrossRefPubMed
  70. 70.↵
    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
    OpenUrlCrossRefPubMedWeb of Science
  71. 71.↵
    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014).
  72. 72.↵
    Priebe, S., Kreisel, C., Horn, F., Guthke, R. & Linde, J. FungiFun2: a comprehensive online resource for systematic analysis of gene lists from fungal species. Bioinformatics 31, 445–446 (2015).
    OpenUrlCrossRefPubMed
  73. 73.↵
    Oberegger, H., Schoeser, M., Zadra, I., Abt, B. & Haas, H. SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulans. Mol Microbiol 41, 1077–1089 (2001).
    OpenUrlCrossRefPubMedWeb of Science
  74. 74.↵
    Hervás-Aguilar, A. & Peñalva, M. A. Endocytic machinery protein SlaB is dispensable for polarity establishment but necessary for polarity maintenance in hyphal tip cells of Aspergillus nidulans. Eukaryot Cell 9, 1504–1518 (2010).
    OpenUrlAbstract/FREE Full Text
  75. 75.↵
    Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012).
    OpenUrlCrossRefPubMedWeb of Science
  76. 76.↵
    Basenko, E. Y. et al. FungiDB: an integrated bioinformatic resource for fungi and Oomycetes. J Fungi 4, 39 (2018).
  77. 77.↵
    Kearse, M. et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).
    OpenUrlCrossRefPubMedWeb of Science
Back to top
Posted November 28, 2024.
Download PDF

Supplementary Material

Email
Cooperative [2Fe-2S] cluster-binding regulates the functional transitions of the Aspergillus fumigatus iron regulator HapX for adaptation to iron starvation, sufficiency and excess
Simon Oberegger, Matthias Misslinger, Hubertus Haas
bioRxiv 2024.11.27.625597; doi: https://doi.org/10.1101/2024.11.27.625597
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area