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

Abstract

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies-many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains-the wild type and a hyper-resistant strain developed through adaptive laboratory evolution-before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi.

Keywords: black yeast; melanin; proteomics; radiobiology; translation.

Figure 1

Resistance of wild type (WT) and Evolved strains to γ-radiation. (A) Survival curve showing the percent of cells recovered for each strain after exposure to increasing doses, up to 4 kGy. (B) Survival of each strain after exposure to a dose of 500 Gy and recovery in fresh medium for 1 h, measured from a subset taken from the same samples that were processed for proteomics.

Figure 2

Proteomic response to γ-radiation exposure in WT and Evolved Exophiala dermatitidis strains. (A) Total number of proteins identified in each condition. * indicates significance (ANOVA, p < 1 × 10⁻⁵). (B) PCA of the total proteomic dataset. Each point represents a biological replicate with color corresponding to condition. (C) Venn diagram depicting overlap of proteins identified in greater than 4 replicates of each condition (>3 for the Evolved control). (D) Heatmap of significantly differentially enriched proteins. Each row represents a single protein and each column represents one replicate, grouped by k-means clustering. Cell colors represent abundance values in comparison to a centered intensity. (E) Volcano plots of pairwise comparisons, with –log₁₀ unadjusted p-values plotted against the log₂ fold change. Black points represent differentially enriched proteins at α = 0.05 after FDR adjustment. (WTc = WT cultures with no radiation exposure; WTr = WT cultures exposed to radiation; WTEc = evolved WT strain with no radiation exposure; WTEr = evolved WT strain exposed to radiation).

Figure 3

Intensity levels of proteins decreased (A) and increased (B) after irradiation in both WT and evolved strains. Error bars represent standard error. Asterisks indicate instances of statistically significant comparisons.

Figure 4

Intensity levels of proteins decreased in the Evolved strains, in comparison with WT strains. Note that only a portion of the Y axis is shown to highlight the small, but significant, differences in intensity levels. Error bars represent standard error. Asterisks indicate instances of statistically significant comparisons.

Figure 5

Intensity levels of non-ribosomal proteins increased in the Evolved strains, in comparison with WT strains. Note that only a portion of the Y axis is shown in the upper panel to highlight the small, but significant, differences in intensity levels. Error bars represent standard error. Asterisks indicate instances of statistically significant comparisons.

Figure 6

Intensity level readings of ribosomal proteins increased in the Evolved strains, in comparison with WT strains. Note that only a portion of the Y axis is shown in the first three panels to highlight the small, but significant, differences in intensity levels. Error bars represent standard error. Asterisks indicate instances of statistically significant comparisons.