The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

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.

ABSTRACTOver the last 3 decades, the frequency of life-threatening human fungal infections has increased as advances in medical therapies, solid-organ and hematopoietic stem cell transplantations, an increasing geriatric population, and HIV infections have resulted in significant rises in susceptible patient populations. Although significant advances have been made in understanding how fungi cause disease, the dynamic microenvironments encountered by fungi during infection and the mechanisms by which they adapt to these microenvironments are not fully understood. As inhibiting and preventingin vivofungal growth are main goals of antifungal therapies, understandingin vivofungal metabolism in these host microenvironments is critical for the improvement of existing therapies or the design of new approaches. In this minireview, we focus on the emerging appreciation that pathogenic fungi likeCandida albicans,Cryptococcus neoformans, andAspergillus fumigatusare exposed to oxygen-limited or hypoxic microenvironments during fungal pathogenesis. The implications of thesein vivohypoxic microenvironments for fungal metabolism and pathogenesis are discussed with an aim toward understanding the potential impact of hypoxia on invasive fungal infection outcomes.

Gliotoxin (1), a major product of the gli non-ribosomal peptide synthetase (NRPS) gene cluster, is strongly associated with virulence of the opportunistic human pathogen Aspergillus fumigatus. Despite identification of the gli cluster, the pathway of gliotoxin biosynthesis has remained elusive, in part because few potential intermediates have been identified. In addition, previous studies suggest that knowledge of gli-dependent metabolites is incomplete. Here we use differential analysis by 2D NMR spectroscopy (DANS) of metabolite extracts derived from gli knock-out and wild-type (WT) strains to obtain a detailed inventory of gli-dependent metabolites. DANS-based comparison of the WT metabolome with that of ΔgliZ, a knock-out strain devoid of the gene encoding the transcriptional regulator of the gli cluster, revealed nine novel gliZ-dependent metabolites including unexpected structural motifs. Their identification provides insight into gliotoxin biosynthesis and may benefit studies of the role of the gli cluster in A. fumigatus virulence. Our study demonstrates the utility of DANS for correlating gene expression and metabolite biosynthesis in microorganisms.

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ⌬dscA and ⌬dscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ⌬dscA and ⌬dscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ⌬dscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.A s mortality due to invasive fungal infections (IFIs) continues to remain high despite improved diagnostics and prophylactic use of antifungal drugs, research to better understand fungal pathogenesis mechanisms is important. The aim of this research is to uncover new aspects of fungal physiology or the fungus-host interaction that can be manipulated to improve treatment outcomes. Investigating the host microenvironments encountered by human-pathogenic fungi during the initiation, development, and active stages of subsequent infection and how fungi adapt to these dynamic environments is one area where potential therapeutic opportunities exist.Recently, there has been an increased interest in how levels of oxygen at the site of infection impact the outcome of IFIs (18, 22). A major reason for this interest is that fungal strains with a deficient ability to adapt to hypoxia fail to cause lethal disease in murine models. Among the best examples of these fungal strains are the Cryptococcus neoformans and Aspergillus fumigatus null mutants of the sterol regulatory element-binding proteins (SREBP) (Sre1 and SrbA, respectively) (11,12,46). SREBPs were first identified in higher eukaryotes as regulators of cholesterol and lipid metabolism (7,19,20,37,45). Mammalian SREBPs are synthesized as endoplasmic reticulum (ER) membrane-bound precursors and contain...

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both

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BackgroundHypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America.Methodology/Principal FindingsIn this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MSE proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses.Conclusions/SignificanceIn this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.