Characterization of heme metabolism and regulation in Aspergillus fumigatus

Iron is essential for numerous cellular processes but toxic in excess. Adaptation to iron starvation is a crucial virulence determinant of pathogens including fungi such as Aspergillus fumigatus. Due to the fundamental differences in the mechanism employed by host and pathogens to maintain iron homeostasis, iron metabolism represents an attractive new target for improvement of antifungal therapy and diagnosis of fungal infections. Iron metabolism is interconnected with heme metabolism. Heme is a central cellular component that is required as co- factor for a variety of cellular processes such as respiration, ergosterol biosynthesis, oxidative stress detoxification, reductive iron assimilation and it is a potential signaling molecule. The current project aims to elucidate heme- dependent regulation at the molecular level in A. fumigatus, the most common airborne fungal pathogen. The studies will include the (i) characterization of the genome-wide transcriptional response to heme depletion, (ii) comparison of the transcriptional response to depletion of heme, iron, and hypoxia, respectively, in wild type and mutant strains involved in these regulatory circuits, (iii) in vivo and in silico promoter analysis of heme-regulated genes, (iv) characterization of heme degradation, and (v) functional analysis of heme-regulated genes. These studies are expected to reveal new regulatory circuits with implication in fungal virulence and drug resistance.

Iron is essential for numerous cellular processes but toxic in excess. Adaptation to iron starvation is a crucial virulence determinant of pathogens including fungi such as Aspergillus fumigatus. Due to the fundamental differences in the mechanism employed by host and pathogens to maintain iron homeostasis, iron metabolism represents an attractive new target for improvement of antifungal therapy and diagnosis of fungal infections. Iron metabolism is interconnected with heme metabolism. Heme is a central cellular component that is required as co-factor for a variety of cellular processes such as respiration, ergosterol biosynthesis, oxidative stress detoxification, reductive iron assimilation and it is a potential signaling molecule. The current project aims to elucidate heme-dependent regulation at the molecular level in A. fumigatus, the most common airborne fungal pathogen. Therefore, we generated a mutant lacking the first enzymatic step of heme biosynthesis (aminolevulinic acid synthase), which allowed to control the cellular heme level by supplementation with aminolevulinic acid (ALA). The growth of this mutant was fully rescued by supplementation with ALA but only partially by heme demonstrating that A. fumigatus has only a limited capacity to utilize exogenous heme. Transcriptional profiling revealed several pathways that are upregulated by heme limitation including mutual-dependent heme and isoprenoid biosynthesis as well as respiration. Upregulation of glycolysis and alcohol metabolism indicated a partial switch from aerobic respiration to fermentation. The downregulation of gene clusters encoding pseurotin A and fumagillin indicated a link between heme and secondary metabolism. In line with the transcriptional profiling data, ALA limitation increased the susceptibility to oxidative stress and iron starvation, as well as to inhibition of ergosterol biosynthesis and respiration. Moreover, we found that prevention of toxic accumulation of protoporphyrine IX, the iron free- precursor of heme during iron starvation is mainly mediated by transcriptional repression of HemA activity by the iron regulatory transcription factor HapX as well as that the SREBPs (sterol regulatory element binding transcription factors) SrbA and SrbB are involved activation of heme biosynthesis. Moreover, this project helped to characterize the roles of fungoglobin and siroheme in A. fumigatus. Fungoglobin is the first identified fungal hemoglobin in the fungal kingdom. It is crucial for adaptation to extreme oxygen depletion and is upregulated by oxygen depletion, iron starvation and heme depletion. Siroheme is a heme-like prosthetic group used by plants and microorganisms, which is an essential cofactor for nitrite and sulfite reductases, but is absent in higher eukaryotes. Lack of siroheme resulted in the inability to utilize sulfate and nitrate as sulfur and nitrogen sources, respectively, and caused significantly decreased resistance against nitric oxide (NO) underlining the importance of nitrite reductase in NO detoxification.