A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin

Abstract

Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity.

Fig. 1.

Growth comparison of BY4743 in YPD or YPG media. The parental strain BY4743 was grown in liquid media supplemented with 2% dextrose (YPD) for 20 h or with 3% glycerol (YPG) for 48 h at 30 °C in the presence of different concentrations of Tcin. The rho⁰ version of BY4743 was grown for 48 h at 30 °C. The experiments were performed in triplicate and repeated twice. Growth is represented by mean OD₆₀₀ values (± SE).

Fig. 2.

Functional classification of yeast deletion mutants that conferred resistance to 4 μM Tcin. (A) Yeast deletion mutants exhibiting resistance to 4 μM Tcin. The figure shows the GO terms (www.yeastgenome.org) most associated with the genes identified. Genes associated with heat shock protein, signal transduction, proteasome/ubiquitination, transcription and RNA metabolism constitute the “Other” category. M/P and Ribo refer to membrane/phospholipid and cytosolic ribosomes. (B) Classification of the mitochondria-associated deletion mutants.

Fig. 3.

Growth of the most resistant 15 deletion strains in the presence of different antibiotics in YPD or YPG media. The Tcin-resistant strains were grown in liquid YPD and YPG media containing (A) 12 μM Tcin, (B) 10 μg/mL anisomycin, or (C) 1 mg/mL chloramphenicol. Relative growth was calculated as the ratio between growth of treated cells to that of untreated cells with a ratio of 1.0, indicating no effect on growth. The experiments were performed in triplicate and repeated twice.

Fig. 4.

Effect of Tcin on mitochondrial translation. (A) Mitochondrial translation rate of the BY4743 cells was determined by measuring the rate of [³⁵S]-methionine incorporation in vivo at increasing concentrations of Tcin. All treatments were performed for 1 h and the rate of translation in the absence of Tcin was normalized to 100%. (B) Autoradiogram of labeled mitochondrial translation products separated on a 15% SDS-polyacrylamide gel. (C) The ratio of translation between Tcin-treated and untreated samples for both total and mitochondrial translation is represented as percent of the untreated control. Each graph represents the mean of four independent experiments with error bars representing the standard deviation.

Fig. 5.

Effect of Tcin on mitochondrial morphology. Mitochondrial morphology of (A) BY4743 control, (B) BY4743 treated with 8 μM Tcin for 4 h, (C) BY4743 treated with 80 μM Tcin for 4 h, (D) fzo1Δ control, and (E) fzo1Δ treated with 8 μM Tcin for 4 h were analyzed using epifluorescence microscopy. Cells were transformed with pVT100U-mtGFP, containing GFP targeted to the mitochondrial matrix. Nuclear and mitochondrial DNA was stained with DAPI. Differential interference contrast (DIC) images of each cell are presented. (Scale bar, 5 μm.)