To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB ^KU80 ). Homologous integration reached about 80% for both calcineurin A ( calA ) and polyketide synthase pksP (alb1 ) genes in the akuB ^KU80 mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB ^KU80 had no influence on pathogenicity in a low-dose murine infection model.

Iron is essential for a wide range of cellular processes. Here we show that the bZIP-type regulator HapX is indispensable for the transcriptional remodeling required for adaption to iron starvation in the opportunistic fungal pathogen Aspergillus fumigatus. HapX represses iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster and heme biosynthesis. In agreement with the impact on mitochondrial metabolism, HapX-deficiency decreases resistance to tetracycline and increases mitochondrial DNA content. Pathways positively affected by HapX include production of the ribotoxin AspF1 and siderophores, which are known virulence determinants. Iron starvation causes a massive remodeling of the amino acid pool and HapX is essential for the coordination of the production of siderophores and their precursor ornithine. Consistent with HapX-function being limited to iron depleted conditions and A. fumigatus facing iron starvation in the host, HapX-deficiency causes significant attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrates that HapX-dependent adaption to conditions of iron starvation is crucial for virulence of A. fumigatus. Due to its requirement for a wide range of cellular processes, iron is an essential nutrient for virtually every organism. The mammalian immune system utilizes iron-withholding mechanisms to deny access to free iron. Therefore, pathogens must overcome extreme iron limitation. Patients with suppressed immune systems due to cancer treatments, organ transplantation, or genetic disorders are at high risk of infection with the ubiquitously present fungal pathogen Aspergillus fumigatus. In this study we found that in Aspergillus fumigatus iron starvation results in drastic metabolic changes depending on the transcription factor HapX. During iron starvation, HapX functions include the repression of iron-consuming pathways to spare iron and activation of iron uptake by siderophores. Siderophores are small molecules able to “steal” iron from host molecules and have previously been shown to play a crucial role in the virulence of Aspergillus fumigatus. Genetic inactivation of HapX attenuates virulence of Aspergillus fumigatus in a murine model of aspergillosis, demonstrating that adaption to iron limitation is a crucial virulence determinant. The identification of numerous HapX-affected genes with a yet uncharacterized link to iron will aid in the further characterization of the metabolic pathways required for fungal adaption to iron starvation and virulence traits.

Summary: Aspergillus fumigatus, the most common airborne fungal pathogen of humans, employs two high‐affinity iron uptake systems: iron uptake mediated by the extracellular siderophore triacetylfusarinine C and reductive iron assimilation. Furthermore, A. fumigatus utilizes two intracellular siderophores, ferricrocin and hydroxyferricrocin, to store iron. Siderophore biosynthesis, which is essential for virulence, is repressed by iron. Here we show that this control is mediated by the GATA factor SreA. During iron‐replete conditions, SreA deficiency partially derepressed synthesis of triacetylfusarinine C and uptake of iron resulting in increased cellular accumulation of both iron and ferricrocin. Genome‐wide DNA microarray analysis identified 49 genes that are repressed by iron in an SreA‐dependent manner. This gene set, termed SreA regulon, includes all known genes involved in iron acquisition, putative novel siderophore biosynthetic genes, and also genes not directly linked to iron metabolism. SreA deficiency also caused upregulation of iron‐dependent and antioxidative pathways, probably due to the increased iron content and iron‐mediated oxidative stress. Consistently, the sreA disruption mutant displayed increased sensitivity to iron, menadion and phleomycin but retained wild‐type virulence in a mouse model. As all detrimental effects of sreA disruption are restricted to iron‐replete conditions these data underscore that A. fumigatus faces iron‐depleted conditions during infection.

Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from l -tyrosine. Proteome analysis indicated that the l -tyrosine degradation enzymes are synthesized when the fungus is grown with l -tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase ( hmgA ) and 4-hydroxyphenylpyruvate dioxygenase ( hppD ). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by l -tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus . Homogentisic acid is the major intermediate, and the l -tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.