The Oxidative Stress Response Highly Depends on Glucose and Iron Availability in Aspergillus fumigatus.

Pathogens have to cope with oxidative, iron- and carbon(glucose)-limitation stresses in the human body. To understand how combined iron-carbon limitation alters oxidative stress responses, Aspergillus fumigatus was cultured in glucose-peptone or peptone containing media supplemented or not with deferiprone as an iron chelator. Changes in the transcriptome in these cultures were recorded after H2O2 treatment. Responses to oxidative stress were highly dependent on the availability of glucose and iron. Out of the 16 stress responsive antioxidative enzyme genes, only the cat2 catalase-peroxidase gene was upregulated in more than two culturing conditions. The transcriptional responses observed in iron metabolism also varied substantially in these cultures. Only extracellular siderophore production appeared important regardless of culturing conditions in oxidative stress protection, while the enhanced synthesis of Fe-S cluster proteins seemed to be crucial for oxidative stress treated iron-limited and fast growing (glucose rich) cultures. Although pathogens and host cells live together in the same place, their culturing conditions (e.g., iron availability or occurrence of oxidative stress) can be different. Therefore, inhibition of a universally important biochemical process, like Fe-S cluster assembly, may selectively inhibit the pathogen growth in vivo and represent a potential target for antifungal therapy.

Functional characterization of genes encoding cadmium pumping P1B-type ATPases in Aspergillus fumigatus and Aspergillus nidulans.

Several P1B-type ATPases are important Cd2+/Cu2+ pumps in Aspergillus species, and they are tightly associated with the heavy metal stress tolerance of these ascomycetous fungi. To better understand the roles of the two P1B-type ATPases, Aspergillus nidulans CrpA Cd2+/Cu2+ pump (orthologue of the Candida albicans Crp1 Cd2+/Cu2+ pump) and Aspergillus fumigatus PcaA Cd2+ pump (orthologue of the Saccharomyces cerevisiae Pca1 Cd2+ pump), we have generated individual mutants and characterized their heavy metal susceptibilities. The deletion of CrpA in A. nidulans has led to the increased sensitivity of the fungus to stresses induced by Zn2+, Fe2+, or the combination of oxidative-stress-inducing menadione sodium bisulfite and Fe3+. Heterologous expression of A. fumigatus PcaA in the S. cerevisiae pca1 deletion mutant has resulted in enhanced tolerance of the yeast to stresses elicited by Cd2+or Zn2+ but not by Fe2+/Fe3+ or Cu2+. Mammalian host immune defense can attack microbes by secreting Zn2+ or Cu2+, and the oxidative stress induced by host immune systems can also disturb metal (Cu2+, Fe2+, and Zn2+) homeostasis in microbes. In summary, PcaA and CrpA can protect fungal cells from these complex stresses that contribute to the virulence of the pathogenic Aspergillus species. Moreover, due to their presence on the fungal cell surface, these P1B-type ATPases may serve as a novel drug target in the future. IMPORTANCE Mammalian host immune defense disrupts heavy metal homeostasis of fungal pathogens. P1B-type ATPase of Aspergillus fumigatus and Aspergillus nidulans may help to cope with this stress and serve as virulence traits. In our experiments, both A. nidulans Cd2+/Cu2+ pump CrpA and A. fumigatus Cd2+ pump PcaA protected fungal cells from toxic Zn2+, and CrpA also decreased Fe2+ susceptibility most likely indirectly. In addition, CrpA protected cells against the combined stress induced by the oxidative stressor menadione and Fe3+. Since P1B-type ATPases are present on the fungal cell surface, these proteins may serve as a novel drug target in the future.

Stress Responses Elicited by Glucose Withdrawal in Aspergillus fumigatus.

Glucose is a widely used carbon source in laboratory practice to culture Aspergillus fumigatus, however, glucose availability is often low in its "natural habitats", including the human body. We used a physiological−transcriptomical approach to reveal differences between A. fumigatus Af293 cultures incubated on glucose, glucose and peptone, peptone (carbon limitation), or without any carbon source (carbon starvation). Autolytic cell wall degradation was upregulated by both carbon starvation and limitation. The importance of autolytic cell wall degradation in the adaptation to carbon stress was also highlighted by approximately 12.4% of the A. fumigatus genomes harboring duplication of genes involved in N-acetyl glucosamine utilization. Glucose withdrawal increased redox imbalance, altered both the transcription of antioxidative enzyme genes and oxidative stress tolerance, and downregulated iron acquisition, but upregulated heme protein genes. Transcriptional activity of the Gliotoxin cluster was low in all experiments, while the Fumagillin cluster showed substantial activity both on glucose and under carbon starvation, and the Hexadehydro-astechrome cluster only on glucose. We concluded that glucose withdrawal substantially modified the physiology of A. fumigatus, including processes that contribute to virulence. This may explain the challenge of predicting the in vivo behavior of A. fumigatus based on data from glucose rich cultures.

Effect of the combinatorial iron-chelation and oxidative stress on the growth of Aspergillus species.

The growth of 14 Aspergillus strains belonging to nine species was studied under combinatorial deferriprone - H2O2 (iron-chelation - oxidative) stress. When deferriprone pretreated mycelia were subjected to even a weak oxidative stress, the growth inhibitory effect of iron-chelation stress was enhanced in 10 out of 14 strains. In contrast, oxidative stress pretreatment of conidia increased their deferriprone tolerance in 10 strains. Applying iron-chelators as antifungal agent or adjuvant can enhance the efficiency of the combinatorial iron withdrawal - oxidative stress strategy of our immune system and may reduce the survival of conidia escaped from the oxidative attack of pulmonary macrophages.

Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans.

Understanding the coordinated regulation of the hundreds of carbohydrate-active enzyme (CAZyme) genes occurring in the genomes of fungi has great practical importance. We recorded genome-wide transcriptional changes of Aspergillus nidulans cultivated on glucose, lactose, or arabinogalactan, as well as under carbon-starved conditions. We determined both carbon-stress-specific changes (weak or no carbon source vs. glucose) and carbon-source-specific changes (one type of culture vs. all other cultures). Many CAZyme genes showed carbon-stress-specific and/or carbon-source-specific upregulation on arabinogalactan (138 and 62 genes, respectively). Besides galactosidase and arabinan-degrading enzyme genes, enrichment of cellulolytic, pectinolytic, mannan, and xylan-degrading enzyme genes was observed. Fewer upregulated genes, 81 and 107 carbon stress specific, and 6 and 16 carbon source specific, were found on lactose and in carbon-starved cultures, respectively. They were enriched only in galactosidase and xylosidase genes on lactose and rhamnogalacturonanase genes in both cultures. Some CAZyme genes (29 genes) showed carbon-source-specific upregulation on glucose, and they were enriched in ß-1,4-glucanase genes. The behavioral ecological background of these characteristics was evaluated to comprehensively organize our knowledge on CAZyme production, which can lead to developing new strategies to produce enzymes for plant cell wall saccharification.

AtfA-Independent Adaptation to the Toxic Heavy Metal Cadmium in Aspergillus nidulans.

Cadmium is an exceptionally toxic industrial and environmental pollutant classified as a human carcinogen. In order to provide insight into how we can keep our environment safe from cadmium contamination and prevent the accumulation of it in the food chain, we aim to elucidate how Aspergillus nidulans, one of the most abundant fungi in soil, survives and handles cadmium stress. As AtfA is the main transcription factor governing stress responses in A. nidulans, we examined genome-wide expression responses of wild-type and the atfA null mutant exposed to CdCl2. Both strains showed up-regulation of the crpA Cu2+/Cd2+ pump gene and AN7729 predicted to encode a putative bis(glutathionato)-cadmium transporter, and transcriptional changes associated with elevated intracellular Cys availability leading to the efficient adaptation to Cd2+. Although the deletion of atfA did not alter the cadmium tolerance of the fungus, the cadmium stress response of the mutant differed from that of a reference strain. Promoter and transcriptional analyses of the "Phospho-relay response regulator" genes suggest that the AtfA-dependent regulation of these genes can be relevant in this phenomenon. We concluded that the regulatory network of A. nidulans has a high flexibility allowing the fungus to adapt efficiently to stress both in the presence and absence of this important transcription factor.

The DUG Pathway Governs Degradation of Intracellular Glutathione in Aspergillus nidulans.

Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses.IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.

General stress response or adaptation to rapid growth in Aspergillus nidulans?

Genome-wide transcriptional changes in Aspergillus nidulans induced by nine different stress conditions were evaluated to reveal the general environmental stress response gene set showing unidirectional expressional changes under various types of stress. Clustering the genes by their transcriptional changes was a useful technique for identifying large groups of co-regulated genes. Altogether, 1642 co-upregulated and 3916 co-downregulated genes were identified. Nevertheless, the co-regulated genes describe the difference between the transcriptomes recorded under the stress conditions tested and one chosen reference culture condition which is designated as the "unstressed" condition. Obviously, the corresponding transcriptional differences may be attributed to either the general stress response or the reference condition. Accordingly, reduced growth and increased transcription of certain antioxidative enzymes observed under stress may be interpreted as elements of the general stress response or as a feature of the "optimal growth" reference condition and decreased antioxidative protection due to "rapid growth" stress. Reversing the many to one comparison underlying the identification of co-regulated gene sets allows the same procedure to highlight changes under a single condition with respect to a set of other "background" conditions. As an example, we compared menadione treatment to our other conditions and identified downregulation of endoplasmic reticulum dependent processes and upregulation of iron-sulfur cluster assembly as well as glutathione-S-transferase genes as changes characteristic of MSB-treated cultures. Deletion of the atfA gene markedly altered the co-regulated gene sets primarily by changing the reference transcriptome; not by changing the stress responsiveness of genes. The functional characterization of AtfA-dependent co-regulated genes demonstrated the involvement of AtfA in the regulation of both vegetative growth and conidiogenesis in untreated cultures. Our data also suggested that the diverse effects of atfA gene deletion on the transcriptome under different stress conditions were the consequence of the altered transcription of several phosphorelay signal transduction system genes, including fphA, nikA, phkA, srrB, srrC, sskA and tcsB. Hopefully, this study will draw further attention to the importance of the proper selection of reference cultures in fungal transcriptomics studies especially when elements of specific stress responses are mapped.

Autolytic hydrolases affect sexual and asexual development of Aspergillus nidulans.

Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40 g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a ß-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.