The Siderophore Ferricrocin Mediates Iron Acquisition in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.

The first promoter for conditional gene expression in Acremonium chrysogenum: iron starvation-inducible mir1(P).

The filamentous fungus Acremonium chrysogenum is of enormous biotechnological importance as it represents the natural producer of the beta-lactam antibiotic cephalosporin C. However, a limitation in genetic tools, e.g. promoters for conditional gene expression, impedes genetic engineering of this fungus. Here we demonstrate that in A. chrysogenum iron starvation induces the production of the extracellular siderophores dimerumic acid, coprogen B, 2-N-methylcoprogen B and dimethylcoprogen as well as expression of the putative siderophore transporter gene, mir1. Moreover, we show that the promoter of mir1, mir1(P), is suitable for conditional expression of target genes in A. chrysogenum as shown by mir1(P)-driven and iron starvation-induced expression of genes encoding green fluorescence protein and phleomycin resistance. The obtained iron-starvation dependent phleomycin resistance indicates the potential use of this promoter for selection marker recycling. Together with easy scorable siderophore production, the co-regulation of mir1 expression and siderophore production facilitates the optimization of the inducing conditions of this expression system.

The interplay between iron and zinc metabolism in Aspergillus fumigatus.

Zinc plays a critical role in a diverse array of biochemical processes. However, excess of zinc is deleterious to cells. Therefore, cells require finely tuned homeostatic mechanisms to balance uptake and storage of zinc. Here we show that iron starvation affects zinc metabolism by downregulating expression of the plasma membrane zinc importer encoding zrfB and upregulating the putative vacuolar zinc transporter-encoding zrcA in Aspergillus fumigatus. Nevertheless, the zinc content of iron-starved mycelia exceeded that of iron replete mycelia, possibly due to unspecific metal uptake induced by iron starvation. In agreement with increased zinc excess and zinc toxicity during iron starvation, deficiency in siderophore-mediated high-affinity iron uptake caused hypersensitivity to zinc. Moreover, an increase of zinc uptake by conditional overexpression of zrfB was more toxic under iron depleted compared to iron replete conditions. This deregulated zinc uptake under iron starvation caused a decrease in heme production and an increase in protoporphyrin IX accumulation. Furthermore, zinc excess impaired production of the extracellular siderophore triacetylfusarinine C but not the intracellular siderophore ferricrocin. Taken together, these data demonstrate a fine tuned coordination of zinc and iron metabolism in A. fumigatus.

EstB-mediated hydrolysis of the siderophore triacetylfusarinine C optimizes iron uptake of Aspergillus fumigatus.

Aspergillus fumigatus excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three N(5)-cis-anhydromevalonyl-N(5)-hydroxy-N(2)-acetyl-l-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene, estB, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of estB in A. fumigatus eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo. Escherichia coli-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three N(2)-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in A. fumigatus.

Interaction of HapX with the CCAAT-binding complex--a novel mechanism of gene regulation by iron.

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.