Additional oxidative stress reroutes the global response of Aspergillus fumigatus to iron depletion

Abstract

Background: Aspergillus fumigatus has to cope with a combination of several stress types while colonizing the human body. A functional interplay between these different stress responses can increase the chances of survival for this opportunistic human pathogen during the invasion of its host. In this study, we shed light on how the H₂O₂-induced oxidative stress response depends on the iron available to this filamentous fungus, using transcriptomic analysis, proteomic profiles, and growth assays.

Results: The applied H₂O₂ treatment, which induced only a negligible stress response in iron-replete cultures, deleteriously affected the fungus under iron deprivation. The majority of stress-induced changes in gene and protein expression was not predictable from data coming from individual stress exposure and was only characteristic for the combination of oxidative stress plus iron deprivation. Our experimental data suggest that the physiological effects of combined stresses and the survival of the fungus highly depend on fragile balances between economization of iron and production of essential iron-containing proteins. One observed strategy was the overproduction of iron-independent antioxidant proteins to combat oxidative stress during iron deprivation, e.g. the upregulation of superoxide dismutase Sod1, the thioredoxin reductase Trr1, and the thioredoxin orthologue Afu5g11320. On the other hand, oxidative stress induction overruled iron deprivation-mediated repression of several genes. In agreement with the gene expression data, growth studies underlined that in A. fumigatus iron deprivation aggravates oxidative stress susceptibility.

Conclusions: Our data demonstrate that studying stress responses under separate single stress conditions is not sufficient to understand how A. fumigatus adapts in a complex and hostile habitat like the human body. The combinatorial stress of iron depletion and hydrogen peroxide caused clear non-additive effects upon the stress response of A. fumigatus. Our data further supported the view that the ability of A. fumigatus to cause diseases in humans strongly depends on its fitness attributes and less on specific virulence factors. In summary, A. fumigatus is able to mount and coordinate complex and efficient responses to combined stresses like iron deprivation plus H₂O₂-induced oxidative stress, which are exploited by immune cells to kill fungal pathogens.

Keywords: Aspergillus fumigatus; Combinatorial stress; Iron deprivation; Oxidative stress; Proteomics; Stress response; Transcriptomics.

Fig. 1

Principal component analysis of the transcriptome data. Symbols represent +Fe/-H₂O₂ (○), +Fe/+H₂O₂ (●), -Fe/-H₂O₂ (□) and -Fe/+H₂O₂ (■) cultures

Fig. 2

Venn-diagrams of the detected changes in the transcriptome and proteome. Graphs indicating the number of up-regulated/down-regulated (a) genes and (b) proteins

Fig. 3

Correlation between proteome and transcriptome data. Data pairs containing the log₂ ratio (log₂FC) of the mean FPKM values (in case of the up- or down regulated genes only) and the log₂ ratio (log₂FC) of the appropriate mean protein abundance values are presented. A - -Fe/-H₂O₂ vs. +Fe/-H₂O₂ B - + Fe/+H₂O₂ vs. +Fe/-H₂O_2. C - -Fe/+H₂O₂ vs. -Fe/-H₂O_2. D - -Fe/+H₂O₂ vs. +Fe/-H₂O_2. E - Pairwise Spearman’s rank correlation coefficients calculated from the log₂FC values (transcriptome vs. proteome). a - all available transcriptome vs. proteome data pairs. b - transcriptome data of up- or down-regulated genes only vs. proteome data. c - proteome data of up- or down-regulated proteins only vs. transcriptome data. n.c. - We found only three up- or down-regulated proteins, therefore a correlation coefficient was not calculated in this case

Fig. 4

DCF production of the –Fe/+H₂O₂ A. fumigatus cultures. Redox imbalance caused by stress treatment was quantified with the 2′,7′-dichlorofluorescin diacetate assay. DCF productions were given as produced pmol DCF / mg dry cell mass (DCM). Mean ± S.D. calculated from three independent experiments are presented. The label “vs. a”, “vs. b”, and “vs. c” represent significantly increased DCF production compared to iron-repleted (+Fe), iron-depleted (-Fe), and H₂O₂-treated iron-replete (+Fe/+H₂O₂) cultures, respectively, according to Student’s t-test (p < 0.05)

Fig. 5

Comparison of oxidative stress response in iron-replete (+Fe/+H₂O₂ vs. +Fe/-H₂O₂) and iron-depleted cultures (-Fe/+H₂O₂ vs. -Fe/-H₂O₂). Percentages of stress responsive genes (differentially expressed genes with at least two-fold transcriptional difference) detected only in iron-depleted cultures (yellow), only in iron-replete cultures (blue), in both cultures with unidirectional transcriptional changes (pink), and in both cultures with opposite transcriptional changes (red) are presented

Fig. 6

The impact of iron supply on oxidative stress resistance of A. fumigatus. A. fumigatus wild type and ΔsidA strains were point-inoculated on minimal medium plates reflecting different iron supply with and without stressors leading to oxidative stress (H₂O₂ and the redox cyclers paraquat and menadione) or BPS, a ferrous iron-specific chelator, which inactivates reductive iron assimilation [30]. Growth was scored after incubation for 48 h at 37 °C. The effect of H₂O₂ in the presence of BPS is not shown because H₂O₂ interferes with BPS function. The A. fumigatus ΔsidA mutant [30] lacks siderophore biosynthesis, which results in decreased iron uptake and decreased resistance to iron starvation. As BPS blocks the growth of ΔsidA [30], this mutant was not analyzed in the presence of BPS. Compared to iron sufficiency (+Fe), iron deprivation (-Fe/BPS) increased the oxidative stress accessibility of the wild type strain (marked by yellow dots); compared to the wild-type strain, SidA-deficiency increased oxidative stress accessibility on +Fe medium (marked by green dots) and -Fe medium (marked by blue dots).

Fig. 7

Simplified model of adaptation mechanism to H₂O₂ stress under iron starvation in A. fumigatus based on the obtained proteomics and transcriptomics data