Beatrix E. Lechner

The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess

Balance of physiological levels of iron is essential for every organism. In Aspergillus fumigatus and other fungal pathogens, the transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron consumption and activating iron uptake. Here, we demonstrate that HapX is also essential for iron resistance via activating vacuolar iron storage. We identified HapX protein domains that are essential for HapX functions during either iron starvation or high‐iron conditions. The evolutionary conservation of these domains indicates their wide‐spread role in iron sensing. We further demonstrate that a HapX homodimer and the CCAAT‐binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter. The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes. Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability.

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.

The interplay between vacuolar and siderophore-mediated iron storage in Aspergillus fumigatus

Iron is an essential element for all eukaryotes but its excess has deleterious effects. Aspergillus fumigatus produces extracellular siderophores for iron uptake and the intracellular siderophore ferricrocin (FC) for distribution and storage of iron. Iron excess has previously been shown to increase the content of ferric FC and the expression of the putative vacuolar iron importer CccA (AFUA_4G12530), indicating a role of both the vacuole and FC in iron detoxification. In this study, we show that CccA-deficiency decreases iron resistance in particular in combination with derepressed iron uptake, while overproduction of CccA increases iron resistance. Green fluorescence protein-tagging confirmed localization of CccA in the vacuolar membrane. In contrast to CccA-deficiency, inactivation of FC biosynthesis did not affect iron resistance, which indicates that vacuolar rather than FC-mediated iron storage is the major iron detoxifying mechanism. After uptake, extracellular siderophore backbones are hydrolyzed and recycled. Lack of FC, CccA, and in particular lack of both increased the cellular content of iron chelated by siderophore breakdown products. These data indicate that the transfer of iron from extracellular siderophores to the metabolism, FC or the vacuole precedes recycling of siderophore breakdown products. Furthermore, this study indicates that CccA does not play an exclusive role in vacuolar iron storage for nutritional reuse.

Perturbations in small molecule synthesis uncovers an iron-responsive secondary metabolite network in Aspergillus fumigatus.

Iron plays a critical role in survival and virulence of the opportunistic pathogen Aspergillus fumigatus. Two transcription factors, the GATA-factor SreA and the bZip-factor HapX oppositely monitor iron homeostasis with HapX activating iron acquisition pathways (e.g., siderophores) and shutting down iron consumptive pathways (and SreA) during iron starvation conditions whereas SreA negatively regulates HapX and corresponding pathways during iron sufficiency. Recently the non-ribosomal peptide, hexadehydroastechrome (HAS; a tryptophan-derived iron (III)-complex), has been found important in A. fumigatus virulence. We found that HAS overproduction caused an iron starvation phenotype, from alteration of siderophore pools to regulation of iron homeostasis gene expression including sreA. Moreover, we uncovered an iron dependent secondary metabolism network where both SreA and HapX oppositely regulate multiple other secondary metabolites including HAS. This circuitry links iron-acquisition and consumption pathways with secondary metabolism—thus placing HAS as part of a metabolic feedback circuitry designed to balance iron pools in the fungus and presenting iron availability as one environmental trigger of secondary metabolism.

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.

Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

Genome mining and functional genomics for siderophore production in Aspergillus niger

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.

Exploiting the Concept of Multivalency with 68Ga- and 89Zr-Labelled Fusarinine C-Minigastrin Bioconjugates for Targeting CCK2R Expression

Cholecystokinin-2 receptors (CCK2R) are overexpressed in a variety of malignant diseases and therefore have gained certain attention for peptide receptor radionuclide imaging. Among extensive approaches to improve pharmacokinetics and metabolic stability of minigastrin (MG) based radioligands, the concept of multivalency for enhanced tumour targeting has not been investigated extensively. We therefore utilized fusarinine C (FSC) as chelating scaffold for novel mono-, di-, and trimeric bioconjugates for targeting CCK2R expression. FSC-based imaging probes were radiolabelled with positron emitting radionuclides (gallium-68 and zirconium-89) and characterized in vitro (log⁡D, IC50, and cell uptake) and in vivo (metabolic stability in BALB/c mice, biodistribution profile, and microPET/CT imaging in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice). Improved targeting did not fully correlate with the grade of multimerization. The divalent probe showed higher receptor affinity and increased CCK2R mediated cell uptake while the trimer remained comparable to the monomer. In vivo biodistribution studies 1 h after administration of the 68Ga-labelled radioligands confirmed this trend, but imaging at late time point (24 h) with 89Zr-labelled counterparts showed a clearly enhanced imaging contrast of the trimeric probe compared to the mono- and dimer. Furthermore, in vivo stability studies showed a higher metabolic stability for multimeric probes compared to the monomeric bioconjugate. In summary, we could show that FSC can be utilized as suitable scaffold for novel mono- and multivalent imaging probes for CCK2R-related malignancies with partly improved targeting properties for multivalent conjugates. The increased tumour accumulation of the trimer 24 h postinjection (p.i.) can be explained by slower clearance and increased metabolic stability of multimeric conjugates.