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19 February 2026

Low-Intensity, Short-Duration Proton Irradiation Enhances Oxidative Stress Sensitivity of Aspergillus nidulans, with Transcriptomic Data Indicating Downregulation of Antioxidative Enzyme Genes

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1
HUN-REN Institute for Nuclear Research (HUN-REN ATOMKI), 4026 Debrecen, Hungary
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Vitrolink LLC., 4033 Debrecen, Hungary
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Department of Molecular Biotechnology and Microbiology, University of Debrecen, 4032 Debrecen, Hungary
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Department of Zoology, Eszterházy Károly Catholic University, 3300 Eger, Hungary
J. Fungi2026, 12(2), 147;https://doi.org/10.3390/jof12020147
This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology
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Abstract

Fungi regularly occur on spacecrafts, posing a serious risk to humans and equipment. In this study, we characterized how the model organism Aspergillus nidulans responds to low-intensity, short-duration proton irradiation designed to simulate a solar particle event, a common stress factor in space. The oxidative stress-sensitive ∆atfA mutant exhibited a lower survival rate than the wild-type strain. Pretreatment of the wild-type strain with menadione sodium bisulfite (MSB), which activates oxidative stress defense mechanisms, increased tolerance to proton beam radiation. These data are consistent with the idea that oxidative defense contributes to cellular responses to ionizing radiation. Unexpectedly, the applied radiation decreased the tolerance to MSB. To understand this unusual behavior, we compared the transcriptomes of the irradiated and non-irradiated mycelia. As expected, proton beam irradiation upregulated many genes involved in DNA repair but downregulated a large number of antioxidant enzyme genes. The downregulation of three key antioxidant genes—prxA (thioredoxin peroxidase), trxB (thioredoxin reductase), and gsh1 (γ-glutamylcysteine synthase)—was further confirmed by RT-qPCR analysis. One possible explanation is that, due to the rapid elimination of reactive oxygen species generated by water radiolysis, the effects of radiolysis-derived electrons could transiently dominate redox signaling. This shift may interfere with redox sensing in the fungus, resulting in reduced antioxidant gene expression and increased sensitivity to oxidative stress. Oxidative stress sensitivity caused by proton radiation may be the Achilles heel of cells that can survive this stress.

1. Introduction

Microorganisms are regular stowaways on spacecrafts, posing a risk to humans [1,2]. They can damage space equipment, potentially infect humans or plants grown in bioregenerative life support systems (BLSSs), and produce allergens and dangerous metabolites (e.g., mycotoxins in BLSSs or health-threatening volatile compounds) [3,4,5,6]. On the other hand, adaptation of the human microbiome to space conditions is vital for the well-being of astronauts [7], and microorganisms are also essential for the efficient functioning of BLSSs [3] or for direct use as raw food material [8]. Furthermore, preventing microbiological contamination of extra-terrestrial environments from Earth is also an emerging problem [9,10].
In addition to extreme vacuum, desiccation, thermal cycling, and microgravity, microorganisms must also cope with different types of ionizing radiation, including γ- and X-rays, ultraviolet C radiation, galactic cosmic rays (GCRs), solar radiation, and trapped radiation when they leave the Earth behind [11,12]. Researchers have intensively studied how microorganisms can survive in these special conditions and how this different environment modifies their behavior (e.g., virulence, resistance to antimicrobial drugs, or secondary metabolite production) [9,13].
Proton radiation (as a major part of GCRs, solar radiation, and trapped radiation in the inner Van Allen belt) can affect microorganisms in low Earth orbit (e.g., aboard the International Space Station). Proton-induced radiation damage to microorganisms is particularly important for planning long-duration crewed missions in deep space to celestial bodies in the Solar System, most importantly to the Moon or Mars [11,12]. Proton irradiation, a form of ionizing radiation, can directly damage organic molecules by disrupting their electronic structures. In aqueous environments, such as within cells, indirect damage may also occur via reactive species generated through proton-induced water radiolysis, including hydroxyl radicals, superoxide anions, hydrogen peroxide, and prehydrated electrons [14]. These direct and indirect effects collectively lead to DNA and protein damage [15,16,17]. Cell survival strongly depends on the efficient repair of DNA double-strand breaks; thus, maintaining antioxidant protection of proteins that preserve the activity of DNA repair enzymes is critical for resilience to ionizing radiation [17]. The effects of proton irradiation on animals, humans, and plants have been extensively studied [18,19,20,21]. In contrast, studies have somewhat ignored the adaptation of fungi to this type of stress and have focused mainly on yeasts [22,23]; however, filamentous fungi pose a risk to astronauts, space equipment, or BLSS plants [3,4,5,6,24].
Despite exposure to radiation and microgravity, the well-controlled environmental conditions aboard the International Space Station (ISS) are conducive to the survival and growth of a wide range of microorganisms, including aspergilli [25,26]. Indeed, Aspergillus species are regularly detected and are often among the most abundant and prevalent fungal taxa in samples collected from spacecraft environments [26,27]. Among aspergilli, Aspergillus nidulans, a ubiquitous filamentous fungus with a global terrestrial distribution, has been repeatedly identified in samples from the ISS [25,26]. Notably, it is one of the few fungal species that have been found in both air and surface samples aboard the station [25]. Furthermore, A. nidulans is a well-established laboratory model organism, making it particularly suitable for investigating microbial adaptation to the unique environmental conditions encountered during spaceflight [28]. Here, we investigated the response of A. nidulans to low-intensity proton beam irradiation. We found that proton irradiation increased the oxidative stress sensitivity of the fungus, and we sought to elucidate the underlying mechanisms responsible for this unexpected effect. The applied radiation (Φ = 1–5 × 1010 p/cm2 with E > 10 MeV) is comparable to the exposure level for many major solar particle events (SPE; Φ > 1010 p/cm2 with E > 10 MeV) [11], representing considerable risk. By providing insights into how life responds to ionizing radiation, our results could benefit astrobiology, human space exploration, and proton therapy.

2. Materials and Methods

2.1. Strains

Aspergillus nidulans THS30.3 (pyrG89, AfupyrG+; pyroA+; veA+) and TNJ92.4 (pyrG89, AfupyrG+; pyroA4; ΔatfA::pyroA; veA+) were studied as a reference strain and a ∆atfA gene deletion mutant, respectively [29]. Strains were maintained on Barratt’s minimal agar plates [30] at 37 °C, and conidia freshly isolated from 5-day-old cultures were used in all experiments.

2.2. Cultures Used for Proton Irradiation Experiments

Conidia were collected using 0.001 v/v % Tween 80 solution and spread (50 µL conidia suspension containing 107 conidia/mL) on Barratt’s minimal agar plates (10 mL in a Petri dish with a diameter of 55 mm) covered with a cellophane sheet. The plates were pre-incubated at 37 °C for 20 h for the THS30.3 strain or for 22 h for the TNJ92.4 strain, which grew more slowly than the THS30.3 strain [29]. These young, still-growing (but not yet conidia-forming) cultures were incubated at 24 °C for 2 h and maintained at 24 °C (for a maximum of 1 h) until irradiation to ensure that the growth status of the cultures remained uniform. After irradiation, the cultures were incubated at 24 °C for two days, and the disk-shaped inhibition zones were examined.
In the combined stress experiments with the THS30.3 strain, either the pre-incubation or post-incubation process was modified. In some cases, after 16 h pre-incubation at 37 °C, mycelia (together with the cellophane sheet) were transferred onto the surface of fresh Barratt’s minimal medium containing 0 or 0.1 mmol/L menadione sodium bisulfite (MSB) as an oxidative stress-generating agent [29] and were further incubated at 37 °C for 4 h until irradiation (control and pre-irradiation stress-treated cultures). In other cases, the irradiated mycelia (together with the cellophane sheet) were transferred onto fresh Barratt’s minimal media containing 0 or 0.1 mmol/L MSB and were incubated at 24 °C for 2 days to observe the disk-shaped inhibition zones (control and post-irradiation stress-treated cultures). Note: Mycelia grown on cellophane sheets and transferred to MSB-free culture medium before or after irradiation behaved essentially the same as those that were not transferred to a new culture medium.
In transcriptome experiments and for detection of reactive oxygen species (ROS), samples were collected 15 min after irradiation from the irradiated zone and from non-irradiated cultures, and were kept at −70 °C until RNA isolation or used immediately for ROS detection.

2.3. Proton Irradiation

Cultures were irradiated at the vertical beamline of the MGC-20E cyclotron located at the Institute for Nuclear Research (HUN-REN ATOMKI) in Debrecen, Hungary (Figure S1A). MGC-20E is a compact isochronous cyclotron. It is a multi-particle accelerator capable of delivering beams of protons, deuterons, and helium ions [31]. The facility has a modernized vacuum and programmable logic controller and a digital NMR field stabilization system, ensuring high reliability for time-critical irradiations. For proton irradiation experiments, the device offers a variable energy range between 2.0 and 18.0 MeV, with a maximum beam current of 50 μA. The beam transport system feeds 4 target vaults with 9 distinct target locations. For experiments requiring precise energy definition, the beam can be passed through an analyzing magnet to significantly reduce the inherent energy spread.
The protons were accelerated to a kinetic energy of 17 MeV and subsequently transported to the irradiation module (Figure S1B) before extraction from the vacuum chamber to air. To achieve quasi-homogeneous irradiation, the proton beam was collimated and defocused, resulting in a 16 mm diameter beam spot positioned off-center within the Petri dishes. All irradiations were conducted with Petri dish lids in place. The protons underwent energy loss as they passed through different layers of media, resulting in an estimated kinetic energy of approximately 13 MeV upon reaching the surface of the biomass. As protons traverse the biomass, a portion of their energy is transferred to the sample. The agar, as the last layer within the layer-stack in the plates, effectively stopped the protons below the biomass (Figure S1C). The irradiation unit constituted a Faraday cup. Accumulated charges of 3200, 6400, and 16,000 ± 20% pC were recorded over an average of 5, 7.5, and 22 min, respectively, for different repeated experiments (n = 3). For samples designated for transcriptome analysis and for detection of ROS, a consistent charge of 6400 ± 20% pC was administered in all cases. Cultures were exposed to protons (1, 2, and 5 × 1010 p/cm2) with energies of E > 10 MeV, mimicking SPE conditions [11].

2.4. High-Throughput RNA Sequencing

Total RNA was isolated from lyophilized mycelia using TRI reagent (Merck Ltd., Budapest, Hungary) according to a previously described method [32]. Six samples (two treatments, untreated and irradiated mycelia, with three biological replicates) were studied. RNA-seq libraries were prepared and sequenced at the Genomic Medicine and Bioinformatics Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. The TruSeq RNA Sample Preparation Kit (Illumina, Praha, Czech Republic) was used for library preparation, according to the manufacturer’s protocol. Library pools were sequenced in one lane of a sequencing flow cell on an Illumina HiScan SQ instrument (Illumina, San Diego, CA, USA) (single-read 75 bp sequencing), and 15–24 million reads per sample were obtained. The reads were aligned to the genome of A. nidulans FGSC A4 (genome: https://fungidb.org/common/downloads/release-65/AnidulansFGSCA4/fasta/data/FungiDB-65_AnidulansFGSCA4_Genome.fasta, accessed on 5 March 2024; gff: https://fungidb.org/common/downloads/release-65/AnidulansFGSCA4/gff/data/FungiDB-65_AnidulansFGSCA4.gff, accessed on 5 March 2024) using hisat2 (version 2.1.0; [33]) (Table S1). Read counts and reads per kilobase million (RPKM) values were generated using FeatureCounts (version 2.0.0; [34]), whereas differentially expressed genes (DEGs) were determined using DESeq2 (version 1.36.0; [35]).

2.5. Evaluation of Transcriptome Data

Upregulated and downregulated genes (irradiation-responsive genes) were defined as DEGs (adjusted p-value < 0.05; DESeq2), where |log2FC| > A. FC (fold change) represents the number calculated with DESeq2 (version 1.36.0) when untreated cultures were used as the reference transcriptome, and A represents the threshold value, which was set to 0.5, 1, or 2.
Gene set enrichment analyses (ShinyGo platform; bioinformatics.sdstate.edu/go/; accessed on 15 August 2025) were used to characterize the upregulated and downregulated gene sets (with thresholds of 0.5, 1, and 2). Default settings were applied, and only biological process GO terms were studied. Hits with a corrected p-value < 0.01 were regarded as significantly enriched.
Enrichment of the following custom gene groups in the upregulated and downregulated gene sets generated with threshold 0.5, 1, or 2 was tested with Fisher’s exact test (p < 0.05; “fisher.test” function of R project; r-project.org/): “Glycolysis”, “Oxidative pentose-phosphate shunt”, “Tricarboxylic acid cycle” (TCA cycle) [36], “Respiration” (Kyoto Encyclopedia of Genes and Genomes pathway database; https://www.kegg.jp/pathway/ani00190; accessed on 15 August 2025), “Antioxidant enzyme”, “Squalene–ergosterol pathway” [37], “Glutathione synthesis, degradation, transport” [38], “Secondary metabolite cluster” [39], “Regulation of oxidative stress response” [40,41], and “Transcription factor” genes (FungiDB; fungidb.org).

2.6. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Assay

RNA was isolated as described in the previous section for RNA sequencing, using independent biological samples. RT-qPCR reactions were performed with the Luna Universal One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Relative transcript levels were calculated using ΔCP values, defined as ΔCP = CPreference gene − CPtarget gene, where CP represents the crossing point of the reaction, and the reference gene was actA, encoding γ-actin. Primer sequences are provided in Table S1.

2.7. Detection of Reactive Oxygen Species (ROS)

Mycelial samples from irradiated and non-irradiated cultures were transferred into 0.5 mL of fresh Barratt’s minimal broth supplemented with 10 μmol/L 2′,7′-dichlorofluorescein diacetate. After 1 h of incubation at 24 °C, the mycelia were harvested by centrifugation (10,000× g, 10 min, 4 °C) and resuspended in 0.5 mL of 5% (w/v) 5′-sulfosalicylic acid solution. The formed 2′,7′-dichlorofluorescin (DCF) was detected spectrofluorimetrically from the cell-free extract as described previously [29]. Results were calculated from five biological replicates and expressed as pmol DCF per mg dry cell mass (DCM), which was determined in parallel experiments.

3. Results

Irradiation experiments were conducted using an MGC-20E cyclotron, which, like most compact medical or experimental cyclotrons, is incapable of stably reproducing the extremely low dose rates of the GCR background. Furthermore, the GCR spectrum includes relativistic heavy ions in the GeV–TeV range, which are beyond the energy reach of MGC-20E. This technical limitation necessitated a focus on SPEs. Therefore, the total collected charges for the exposures were set at 3200, 6400, and 16,000 pC (±20%), which correspond to 1.0 × 1010 p/cm2, 2.0 × 1010 p/cm2, and 5.0 × 1010 p/cm2, respectively. These fluences are in line with the definition of major SPE as an event exceeding a fluence of 1.0 × 1010 proton/cm2 (>10 MeV) [11].
The treatment with a 16,000 pC dose completely killed the wild-type (THS30.3) strain, resulting in a clear zone of inhibition in the treated area (Figure 1a). However, it could survive the 3200 and 6400 pC doses (Figure 1b,c). These treatments resulted in only minor, yet visible, alterations in colony morphology, which can be attributed to delayed growth and conidiation within the irradiated zone. The oxidative stress-sensitive ∆atfA [29] deletion mutant showed elevated sensitivity (particularly at 6400 pC) to proton irradiation (Figure 1d–f).
Figure 1. Proton irradiation tolerance of A. nidulans THS30.3 and TNJ92.4 strains. Cultures were irradiated with accumulated charges of 16,000, 6400, or 3200 pC (over an average of 22, 7.5, and 5 min, respectively). Some cultures were also treated with oxidative stress elicited by 0.1 mmol/L MSB applied before irradiation or after irradiation. After irradiation, the cultures were incubated at 24 °C for 2 days. Representative photos of three biological replicates taken after two days of post-irradiation incubation are shown: (a–c) cultures of the reference strain; (d–f) the ΔatfA mutant; (g–i) the MSB-pretreated cultures (note that cultures irradiated with 3200 pC were photographed at the end of day 1 to demonstrate the weak effect of irradiation, which was no longer visible after 2 days); and (j–l) the cultures treated with MSB after irradiation. The diameter of the Petri dish was 55 mm. The arrows indicate the irradiation zone (where the zone is not apparent).
Mild MSB pretreatment (which upregulates oxidative stress protection [42]) decreased the sensitivity of the THS30.3 reference strain to proton radiation stress; it survived even the 16,000 pC dose (Figure 1g–i). The cross-protection between oxidative and proton radiation stress was asymmetric; proton radiation decreased the tolerance of the reference strain to MSB-induced oxidative stress (Figure 1j–l).
To elucidate this unexpected behavior, we compared the transcription of antioxidative enzyme genes between proton-irradiated and non-irradiated mycelia. Because of the limited size of the samples (the diameter of the irradiated zone was only 16 mm; Figure 1 and Figure S1), we used high-throughput RNA sequencing, which allowed us to evaluate the stress response induced via proton radiation at the transcriptome level, in addition to examining the transcription of antioxidant genes. For these experiments, irradiation with 6400 pC of accumulated charges (2.0 × 1010 p/cm2) was selected for treatment, since the lower dose had too small an effect on the growth of the cultures, while the higher dose completely killed the fungus in the irradiation zone (Figure 1a–c). Note that this fluence can be considered realistic, given that the benchmark “worst-case” SPE used in spacecraft shielding design reached a fluence of 2.25 × 1010 protons/cm2 during the August 1972 event [43]. The time of sample collection was set to 15 min after treatment to focus on the direct/primary consequences of radiation.
The stress treatment caused substantial changes in the transcriptome by affecting the transcription of thousands of genes (Figure 2, Table S1).
Figure 2. Transcriptomic consequences of proton irradiation on A. nidulans (THS30.3). Cultures were irradiated with 6400 pC accumulated charges, and samples were collected 15 min after treatment. The number of upregulated and downregulated genes (DEGs where |log2FC| was higher than 0.5, 1, or 2) is presented.
Gene set enrichment analyses of the upregulated and downregulated gene sets revealed the following:
Genes related to DNA damage stress (e.g., those involved in DNA repair, signal transduction in response to DNA damage, and DNA integrity checkpoint signaling during mitosis) were enriched in the upregulated gene sets (Table 1 and Table S2). DNA damage is a common consequence of ionizing radiation, particularly proton irradiation, which can induce a wide spectrum of DNA damage, including base modifications, single- and double-strand breaks, and complex clustered lesions both directly and indirectly through reactive species generated by radiolysis. Consequently, a broad upregulation of DNA repair genes is frequently observed in response to proton irradiation-induced stress [22,23].
Table 1. The results of the gene set enrichment analysis of the radiation stress-responsive downregulated and upregulated genes.
Genes involved in RNA synthesis (transcription) and ribosome biogenesis (translation) were also enriched in the upregulated gene sets (Table 1 and Table S2). However, the effects of low-intensity ionizing radiation on protein synthesis remain controversial. The upregulation and downregulation of ribosome biogenesis have been observed in various studies [44,45,46]. Downregulation can generally be explained by the high-energy cost of protein synthesis. Conversely, upregulation can be explained by the increased production of proteins important in the DNA damage stress response or by overcompensating for the decrease in ribosome biogenesis caused by nucleolar stress induced by DNA damage [44,45,46,47,48].
Genes involved in primary metabolism, including glucose import, glycolysis, the oxidative pentose-phosphate shunt, pyruvate metabolism, cytochrome c oxidase activity, and ergosterol biosynthesis, were enriched in the downregulated gene sets (Table 1, Table 2 and Tables S2 and S3), which is typical for cultures with reduced growth [37,38,42,49,50]. The treatment also altered the activity of several transcription factor genes that participate in the regulation of developmental processes (conidium and ascospore formation) (Table 2 and Table S3). For example, flbB, flbC, flbD, nosA, and rosA [51,52] were downregulated, whereas stuA and nsdC [51,52] were upregulated (Table S3). These transcriptional changes are in line with the delayed growth and conidiogenesis observed in irradiated cultures, leading to the appearance of a visible irradiation zone (Figure 1).
Table 2. Summary of the transcriptional behavior of selected gene groups.
Stress usually alters the transcription of secondary metabolite cluster genes: some clusters are upregulated, whereas others are downregulated after treatment [29,37,38,49]. This phenomenon was also observed after the proton beam irradiation (Table 1, Table 2 and Tables S2 and S3). Genes of the Sterigmatocystin cluster, the best-known mycotoxin cluster of A. nidulans [53], were enriched in the upregulated gene set (Table 2 and Table S3).
Although the aforementioned changes represent common cellular responses to ionizing radiation, the transcriptional activity of genes encoding antioxidant enzymes showed an unexpected pattern. Genes encoding superoxide dismutases, catalases, heme peroxidases, and components of the thioredoxin/glutaredoxin/glutathione system were predominantly downregulated. Many of these genes exhibited strong downregulation (log2FC < −1) (Figure 3, Table S3) and were significantly enriched among the downregulated gene sets (Table 2 and Table S3). Although genes belonging to the “glutathione synthesis, degradation, and transport” functional category were not enriched in the downregulated gene sets, both genes responsible for glutathione biosynthesis (gsh1 and gsh2) were significantly downregulated (log2FC < −1) (Figure 3; Table S3). Moreover, gsdA and gndA, encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively—enzymes essential for NADPH production required for proper functioning of the thioredoxin/glutaredoxin/glutathione system—were also downregulated (log2FC < −1) (Figure 3; Table S3). Importantly, for the three genes tested (trxB, prxA, and gsh1, encoding thioredoxin reductase, thioredoxin peroxidase, and γ-glutamylcysteine synthetase, respectively), the RT-qPCR data also confirmed the irradiation-initiated downregulation (Figure 3c).
Figure 3. Transcriptional activity of genes encoding antioxidative enzymes involved in glutathione synthesis or NADPH production. Panels a and b: RPKM values (mean ± S.D.; n = 3) are shown to visualize the abundance of appropriate mRNAs in the transcriptome of untreated (blue) and proton beam-irradiated (brown) cultures. Genes with low (a) and high (b) transcriptional activity are shown separately for clarity. On panel a: sodM, AN1131 (superoxide dismutases); catC, AN8553 (catalases); AN5440 (cytochrome c peroxidase); AN7924 (further heme peroxidase); trxB, AN4023, AN5232, AN4304, AN7567, AN9464 (elements of the thioredoxin–glutaredoxin system); gpxA, AN0629 (glutathione peroxidases); glrA (glutathione reductase); gsh1, gsh2 (γ-glutamylcysteine synthetase and glutathione synthase). On panel b: sodA, sodB (superoxide dismutases); catA, catB, catD (catalases); ccp1 (cytochrome c peroxidase); AN2578 (further heme peroxidase); trxA, trxR, trxB, tpxA, prxA, AN8080, AN4215 (elements of the thioredoxin–glutaredoxin system); gsdA, gndA (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) (for further details, see Table S3). ***, **, *—Differentially expressed genes (according to the DESeq2 software 1.51.6) where the log2FC value for the transcriptional changes caused by the treatment was smaller than −2, −1, or −0.5, respectively. Panel (c): RT-qPCR data (mean ± S.D.; n = 3) of selected genes (trxB, prxA and gsh1). The p-values were calculated with Student’s t-test (n = 3).
No general upregulation or downregulation of “Regulation of oxidative stress response” genes was observed (Table S3). The phkB, hk-8-1, hk-8-2, and hk-8-3 genes—components of the two-component signaling system [40]—as well as the atfA transcription factor gene [29] were downregulated (Table S3). In contrast, the genes encoding the RsrA transcription factor, a negative regulator of the oxidative stress response [41]; the NapA transcription factor, a positive regulator of the oxidative stress response [54]; the SskB mitogen-activated protein kinase kinase kinase, an upstream regulator of AtfA [40]; and the histidine kinase hk-8-6 [40] were upregulated (Table S3).
Importantly, proton irradiation induced only a modest and statistically non-significant increase in ROS production, as measured by the DCF assay. Proton-irradiated cultures showed 2.01 ± 0.73 pmol DCF mg−1 DCM, compared with 1.28 ± 0.29 pmol DCF mg−1 DCM in untreated controls (Student’s t-test, p = 0.0715, n = 5). The relatively high variability observed among irradiated samples may reflect the transient and dynamic nature of radiation-induced ROS generation. Given the rapid turnover and scavenging of ROS in fungal cells, short-lived fluctuations in ROS levels could contribute to the observed dispersion and the lack of statistical significance at the examined time point.

4. Discussion

Microbes are common stowaways in space vehicles and stations, where proton radiation (e.g., as part of SPEs or GCR) regularly occurs. This radiation, together with other factors, may decrease their beneficial activity (e.g., raw food material production and plant growth-promoting activity in BLSS) or potentially increase their harmful properties (e.g., production of mycotoxins and allergens that cause human infections) [28]. In addition, proton beam irradiation is routinely used in radiotherapy [55,56], and a deeper understanding of its effects on cells may help further improve its benefits [57,58].
Many microorganisms can survive ionizing radiation, and some, such as Deinococcus radiodurans [59,60], are extremely radiation-resistant. It is assumed that extreme radiation resistance evolved as a by-product of adaptation to desiccation, rather than as a direct consequence of exposure to ionizing radiation [60,61]. In D. radiodurans, efficient and unique DNA repair, strong protection against oxidative stress, redundancy of DNA repair and antioxidant protein genes, and a unique cell wall have been shown to be important for coping with ionizing radiation [60,61]. Moeller et al. (2012) [62] found that efficient DNA repair, the presence of dipicolinic acid and special proteins (α/β-type small, acid-soluble spore proteins), and reduced water content, but not pigmentation, helped the spores of Bacillus subtilis (a model organism in astro- and radiobiology) survive high-energy proton radiation. In the case of Saccharomyces cerevisiae, transcriptional changes recorded after proton irradiation stress suggested protein toxicity stress, DNA damage, and attenuation of cell division: Genes related to “cellular response to DNA damage stimulus”, “DNA biosynthetic processes”, “protein refolding”, and “protein targeting to the mitochondria” were enriched in the upregulated gene set, while genes related to “chromatin assembly or disassembly” were enriched in the downregulated gene set [23]. Schultzhaus et al. (2021) found that melanin pigments had no impact on the proton beam tolerance of Exophiala dermatitidis [22]. Proton irradiation upregulated DNA repair, autophagy, and protein catabolism genes, and downregulated translation and ribosomal biogenesis genes in this fungus [22]. In the case of A. nidulans, as with other studied organisms, the most characteristic response to proton irradiation was the bulk upregulation of DNA repair genes (Table 1 and Table S2).
A stressor can modify the behavior of microbes and consequently their tolerance to other stressors, which is usually observed as cross-protection; that is, adaptation to stress usually increases resistance to forthcoming stress. One explanation for this phenomenon is that different stressors have similar physiological consequences, triggering the same responses under different stresses (e.g., growth reduction, removal of accumulated ROS, breakdown of damaged proteins, and DNA repair) [63,64]. Cross-protection was also evident in our study, as pretreatment with MSB-induced oxidative stress enhanced tolerance to proton radiation (Figure 1g–i). Proton radiation typically generates ROS, and a robust antioxidative defense can mitigate the detrimental effects of these compounds [15,16,17,65,66]. Since previous work has shown that MSB treatment upregulates antioxidant defenses in A. nidulans [29,50], it is plausible that the antioxidative mechanisms activated through MSB pretreatment contributed to the reduced sensitivity of the fungus to proton radiation. This interpretation is consistent with our observation that the oxidative stress-sensitive ∆atfA mutant exhibited increased radiosensitivity (Figure 1d–f). Interestingly, this cross-protection was asymmetric. The proton irradiation applied in this study decreased oxidative stress tolerance (Figure 1j–l). According to our transcriptome and RT-qPCR data, this could be the consequence of the applied low-intensity, short-duration proton irradiation, which downregulated several genes important in antioxidative defense: genes encoding antioxidative enzymes as well as enzymes of glutathione biosynthesis and NADPH production (Figure 3, Table S3).
Proton irradiation can directly damage organic compounds by altering or displacing their electronic structures. Additionally, in environments where these compounds are embedded in water, they may also suffer indirect damage from reactive species produced by the proton-induced radiolysis of water [14]. After excitation, water forms hydroxide radicals (OH·), hydrogen radicals (H·), oxygen atoms (which can produce H2O2 with water), and hydrogen (H2). The ionization of water leads to the formation of water radical cations (H2O·+) and ejected secondary electrons (e−). H2O·+ can form OH· and hydronium ions (H3O+) with a water molecule, whereas as secondary electrons slow down, they form reactive prehydrated electrons and then less reactive hydrated electrons (e−(aq)). Reeves and Kanai (2017) [67] suggested that, during proton beam irradiation, water ionization is more substantial than water excitation, resulting in the formation of large amounts of reactive prehydrated electrons. This is particularly interesting because in this manner, proton irradiation can elicit not only oxidative damage to organic compounds (via ROS) but also reductive damage via reactive prehydrated electrons [67]. In fact, researchers have suggested that reductive DNA damage caused by prehydrated electrons can be even stronger than oxidative damage [68,69,70]. Taken together, our results suggest the following mechanism. Proton irradiation induces water radiolysis, leading to the formation of ROS and prehydrated electrons. A portion of the ROS is rapidly neutralized by the cellular antioxidant defense system, whereas the remaining fraction causes cellular damage. The extent of this damage can be mitigated by pre-activation of the antioxidant defense through MSB pretreatment (Figure 1g–i) or exacerbated by inactivation of the AtfA transcription factor, which is essential for the proper regulation of antioxidant responses (Figure 1d–f). The generated prehydrated electrons may also contribute to cellular damage. However, their elimination appears to be slower than the rapid detoxification of ROS, resulting in a transient state in which reductive processes predominate. This reductive shift is associated with the downregulation of antioxidant enzyme production (Figure 3), thereby increasing cellular sensitivity to oxidative stress during subsequent MSB treatment (Figure 1j–l).
While no general upregulation or downregulation of “Regulation of oxidative stress response” genes was observed, the upregulation of rsrA was notable (Table S3). RsrA is a conserved C2H2 transcription factor that acts as a negative regulator of the oxidative stress response by antagonizing NapA, the major activator of oxidative stress response genes [41]. The regulatory mechanisms controlling rsrA expression and RsrA activity remain largely unknown. Sensing oxidative (or reductive) stress frequently relies on the formation or reduction of disulfide bonds in regulatory proteins, as demonstrated for NapA and the AnCF complex [54,71]. Because electrons generated during radiolysis are capable of cleaving disulfide bonds [72], ionizing radiation may have the potential to transiently downregulate the oxidative stress response by inactivating positive regulators and/or activating negative regulators of oxidative stress response genes.
Further studies are needed to clarify how the occurrence and longevity of this effect depend on the applied irradiation, the type and properties of the irradiated cells, as well as on other stresses occurring concomitantly, particularly microgravity [73]. However, confused redox regulation may represent a type of Achilles heel of proton-irradiated cells that can be used in practice. Proton beam therapy is an important part of cancer radiation therapy [56]. If the high-energy protons (70–250 MeV) used in proton therapy are able to downregulate the antioxidative defenses of human cells, the efficacy of the treatment could potentially be enhanced by combining proton irradiation with subsequent administration of chemotherapeutics, such as doxorubicin [74], which induce oxidative stress.

5. Conclusions

The effects of low-intensity, short-duration proton beam irradiation on A. nidulans were investigated. The observed asymmetric cross-protection between MSB-induced oxidative stress and proton radiation—where MSB pretreatment increased tolerance to proton irradiation, whereas proton irradiation reduced tolerance to MSB—corroborates previous reports [67,70]. This asymmetry indicates that proton radiation induces complex cellular alterations that cannot be attributed solely to ROS formation. We propose that highly reactive prehydrated electrons generated during proton irradiation contribute to the distinctive features of the proton stress response. Electrons arising from water radiolysis, particularly prehydrated electrons, are known to cause reductive damage to macromolecules, including DNA [70,75], and to cleave disulfide bridges [72]. If ROS produced during irradiation are rapidly eliminated, the transient predominance of radiolysis-derived electrons in redox processes may disrupt cellular redox sensing and regulatory networks. Such a shift could lead to the unexpected downregulation of genes encoding antioxidant enzymes and thereby increase oxidative stress sensitivity. Although the duration of this downregulation remains unknown, even a transient reduction in oxidative stress tolerance may have important implications. These findings are relevant both for spacecraft microbial risk management and for the therapeutic application of proton radiation. Further studies are needed to elucidate the molecular mechanisms underlying proton radiation-induced oxidative stress sensitivity, which may finally allow us to benefit from this unexpected consequence of proton irradiation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof12020147/s1, Table S1 (Table S1.xlsx) Alignment statistics, and principal component analysis (PCA) of the RNA sequencing data. Table S2 (Table S2.xlsx) Results of the gene set enrichment analyses. Table S3 (Table S3.xlsx) Transcriptional data for selected gene groups. Figure S1 (Figure S1.pdf) Experimental setup for proton irradiation.

Author Contributions

M.S., T.E. and I.P. conceptualization; M.S., A.F., K.A., Z.S. and T.E. methodology; M.S., I.V., A.F., B.C.G., K.A. and T.E. investigation; M.S., K.A., Z.S. and T.E. writing—original draft preparation; M.S., I.V., A.F., K.A., Z.S., I.P. and T.E. writing—review and editing; I.V., K.A. and T.E. visualization; Z.S., T.E. and I.P. funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Research, Development and Innovation Office (Hungary) project K131767. Project no. TKP2021-EGA-20 and TKP2021-NKTA-42 were implemented with support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development, and Innovation Fund, financed under the TKP2021-EGA and TKP2021-NKTA funding schemes. This research was supported in part by the GINOP-2.3.3-15-2016-00029 ‘HSLab’ project and has received funding from the HUN-REN Hungarian Research Network.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The transcriptome datasets are available in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accessed on 31 January 2026) under accession number GSE294405.

Conflicts of Interest

Author Máté Szarka was employed by the company Vitrolink LL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BLSSBioregenerative life support system
DCMDry cell mass
GCRGalactic cosmic rays
ISSInternational Space Station
MSBMenadione sodium bisulfite
NRPSNonribosomal peptide synthetase
PKSPolyketide synthase
ROSReactive oxygen species
RPKMReads per kilobase million
RT-qPCRReverse transcription-quantitative polymerase chain reaction
SPESolar particle event
TCA cycleTricarboxylic acid cycle

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