The transcriptomic and phenotypic response of the melanized yeast Exophiala dermatitidis to ionizing particle exposure

Abstract

Fungi can tolerate extremely high doses of ionizing radiation compared with most other eukaryotes, a phenomenon encompassing both the recovery from acute exposure and the growth of melanized fungi in chronically contaminated environments. This observation has led to the use of fungi in radiobiology studies, with the goal of finding novel resistance mechanisms that could assist fields such as nuclear medicine and space biology. As photonic (x-ray and γ-ray) exposure is the most widely available method for irradiation, little work has been done examining how fungi (other than budding yeast) respond to irradiation by ionizing particles (e.g. protons), although particle irradiation may cause distinct cellular damage, and it is more relevant to environmental exposure profiles. Here, we expand on a previous study of the melanized yeast Exophiala dermatitidis responding to γ-radiation exposure by characterizing the phenotypic and transcriptomic response to irradiation by three different ionizing particles: protons, deuterons, and α-particles. No significant difference in survival was observed between these strains under any condition, suggesting that melanin does not impart protection to acute irradiation to these particles. The transcriptomic response during recovery to particle exposure was similar to that observed after γ-irradiation, with DNA repair and replication genes upregulated, and genes involved in translation and ribosomal biogenesis being heavily repressed, indicating an attenuation of cell growth. However, a comparison of global gene expression showed clear clustering into particle and γ-radiation groups. The response elicited by particle irradiation was more complex. Compared to the γ-associated response, particle irradiation resulted in greater changes in gene expression, a more diverse set of differentially expressed genes, and a significant induction of gene categories such as autophagy and protein catabolism. Additionally, individual particle responses were analyzed and compared, resulting in the discovery of unique expression signatures and individual genes for each particle type that could be used as radio-nuclide discrimination markers. The strong response we observed here at the gene expression level inprovides insights into the unique types of damage that particle irradiation causes to fungal cells, allowing for future dissection of radiation source-specific resistance mechanisms.

Proteomics Reveals Distinct Changes Associated with Increased Gamma Radiation Resistance in the Black Yeast Exophiala dermatitidis

“…These changes may indicate again a subtle change in the metabolism of this strain, although we did not observe it. Second, several proteins involved in DNA replication and nucleic acid processing, which we observed to be highly induced after irradiation in our previous transcriptomic studies [ 9 , 41 ], were observed at a much higher intensity in the Evolved strains, including Replication factor A2, a Replication protein A subunit (single-stranded DNA binding during repair and replication), ribonucleoside-diphosphate reductase (deoxyribonucleotide synthesis) and a DNA and an RNA helicase. This is significant due to the role that replication plays in DNA repair, and may suggest that Evolved cells have increased concentrations of limiting DNA repair proteins present during exposure, allowing them to recover more efficiently through better processing of damaged DNA sites during replication or other repair processes [ 60 ].…”

“…A transcriptomic and functional genetic analysis of Deinococcus radiodurans produced the same result [ 65 ]. Moreover, induction of RAD52 after irradiation is observed in S. cerevisiae [ 77 ] and in E. dermatitidis [ 9 , 41 ], but the regulation of its expression, or of RAD54 , do not appear to be required for them to carry out their normal functions [ 55 , 78 ], and the transcriptomic response (e.g., induction of DNA repair proteins, inhibition of growth) is either largely similar across many organisms of varying sensitivity to γ-radiation [ 66 , 77 , 79 , 80 , 81 ], or has been shown not to assist in the identification of DNA repair genes at all [ 25 ].…”

“…One is that there is a substantial delay in protein synthesis compared to transcription (i.e., past the 1 h recovery period), so protein production occurs at later time points than our recovery period covered. Because acute ionizing radiation exposure causes extensive oxidative stress, and protein synthesis (including changes in proteomic composition) can occur at time scales much lower than 1 h [ 9 , 41 , 91 ], we typically consider this length of time to be ample to observe changes at any level that can be scrutinized by current -omic technologies. However, the surprisingly small number of changes that we observed here suggests that a more exhaustive and extended time course should be explored in the future with regard to any proteomic response in this organism, and particularly with the response to γ-radiation exposure, as there is evidence in other fungi of different patterns of regulation over time, upon irradiation [ 62 , 77 ].…”

“…Analysis of the proteins that were changed in relative amounts for each strain and condition was performed to identify enriched gene sets in the following manner (and as described for transcriptomics previously [ 9 , 41 ]): Using FungiFun2 ( [ 42 , 43 ]), the relevant lists of gene names were entered and searched for Gene Ontology—Biological Process categories that were significantly enriched using a Fisher’s exact test and an FDR cutoff of <0.01. This provided us with proteins that were overrepresented in the lists of those that were significantly changed in intensity or relative amount under certain strains or conditions.…”

Adaptive evolution of a melanized fungus reveals robust augmentation of radiation resistance by abrogating non‐homologous end‐joining

“…Ionizing radiation (IR) exposure presents a singular type of biological stress, generating various lesions in DNA, including double strand breaks (DSBs), which result in cell death if left unrepaired (Ward, 1988; Iliakis, 1991). Nevertheless, many organisms have demonstrated the ability to recover from extremely high doses of IR, such that could be expected to induce hundreds of DSBs within the genome (Zhdanova et al ., 2000; Liu et al ., 2003; Ingemar et al ., 2005; Rainey et al ., 2005; Dadachova and Casadevall, 2008; Gladyshev and Meselson, 2008; Vaisnav et al ., 2014; Pacelli et al ., 2017; Schultzhaus et al ., 2019, 2020). Fungi in particular have attracted interest due to their generally high resistance to acute IR exposure (Shuryak et al ., 2017; Schultzhaus et al ., 2019; Shuryak, 2019), their apparent increased abundance in environments that are highly contaminated with IR, such as Chernobyl (Zhdanova et al ., 2000) and Fukushima (Shinohara et al ., 2017, 2018), and their ability to sequester radionuclides (White and Gadd, 1990; Haselwandter and Berreck, 1994; Steiner et al ., 2002; Dighton, 2019).…”