Actin cytoskeletal inhibitor 19,20-epoxycytochalasin Q sensitizes yeast cells lacking ERG6 through actin-targeting and secondarily through disruption of lipid homeostasis

Abstract

Repetitive uses of antifungals result in a worldwide crisis of drug resistance; therefore, natural fungicides with minimal side-effects are currently sought after. This study aimed to investigate antifungal property of 19, 20-epoxycytochalasin Q (ECQ), derived from medicinal mushroom Xylaria sp. BCC 1067 of tropical forests. In a model yeast Saccharomyces cerevisiae, ECQ is more toxic in the erg6∆ strain, which has previously been shown to allow higher uptake of many hydrophilic toxins. We selected one pathway to study the effects of ECQ at very high levels on transcription: the ergosterol biosynthesis pathway, which is unlikely to be the primary target of ECQ. Ergosterol serves many functions that cholesterol does in human cells. ECQ's transcriptional effects were correlated with altered sterol and triacylglycerol levels. In the ECQ-treated Δerg6 strain, which presumably takes up far more ECQ than the wild-type strain, there was cell rupture. Increased actin aggregation and lipid droplets assembly were also found in the erg6∆ mutant. Thereby, ECQ is suggested to sensitize yeast cells lacking ERG6 through actin-targeting and consequently but not primarily led to disruption of lipid homeostasis. Investigation of cytochalasins may provide valuable insight with potential biopharmaceutical applications in treatments of fungal infection, cancer or metabolic disorder.

Figure 1

Susceptibility and survival of S. cerevisiae strains with a deletion or overexpression in the gene for the ergosterol biosynthesis during the treatments with different concentrations of fluconazole (a, b), Xylaria extract (c, d), or ECQ (e–h) using micro-dilution assays and spot tests, respectively. Cells from micro-dilution assays were directly spotted (10⁰ dilution) or diluted 1000 times (10^–3 dilution) prior to be spotted on YPD plates. Growth of the overexpression strains were compared with the wild-type strain. * was referred to the mean difference with significant p value of < 0.05.

Figure 2

Relative expression of genes in ergosterol biosynthesis. S. cerevisiae BY4742 wild-type strain was treated with 4 µg/ml of ketoconazole, 500 µg/ml of Xylaria extract, or 500 µg/ml ECQ for 2 h. The relative mRNA levels of the treated cells were compared to the untreated cells and normalized with a housekeeping gene TDH3. At least two independent qRT-PCR experiments were performed in triplicates. Error bars represent standard error of the mean (SEM).

Figure 3

Effect of Xylaria extract and ECQ on actin cytoskeleton organization (a) and cell membrane integrity (b) in the wild-type S. cerevisiae and the ∆erg6 strains. Blue arrows indicated cortical actin patches; red arrows indicated actin fibre; yellow arrows indicated actin body; and orange arrows indicated cell breakage. Error bars represent standard error of the mean (SEM) (*p < 0.05, using one-way ANOVA compared to the untreated condition).

Figure 3

Effect of Xylaria extract and ECQ on actin cytoskeleton organization (a) and cell membrane integrity (b) in the wild-type S. cerevisiae and the ∆erg6 strains. Blue arrows indicated cortical actin patches; red arrows indicated actin fibre; yellow arrows indicated actin body; and orange arrows indicated cell breakage. Error bars represent standard error of the mean (SEM) (*p < 0.05, using one-way ANOVA compared to the untreated condition).

Figure 4

Alteration of sterol and TAG levels as well as formation of LDs clustering were examined during the ECQ treatment in S. cerevisiae. (a) ergosterol biosynthesis pathway in S. cerevisiae, including involved transcriptional factors and targets of antifungal drugs was depicted (b) percentage of identified sterol composition as quantified by GC–MS, (c) LDs formation and Nile Red fluorescent intensity, and the chromatograms of the change in (d) sterol composition and (e) TAG content of yeast cells after treatment with ECQ of S. cerevisiae wild-type and ∆erg6 strains treated with the Xylaria extract or ECQ. Gene labelled in green or red colour indicated up- or down-regulated expression, respectively. Asterisks indicated target of Morpholines. Red arrows indicated increased or decreased accumulation of sterol level, following the ECQ treatment. “ND.” Was referred to “not detected”. Error bars represent standard error of the mean (SEM). Different letters above the error bars (a–f) indicate significant differences at p value of < 0.05.

Figure 4

Alteration of sterol and TAG levels as well as formation of LDs clustering were examined during the ECQ treatment in S. cerevisiae. (a) ergosterol biosynthesis pathway in S. cerevisiae, including involved transcriptional factors and targets of antifungal drugs was depicted (b) percentage of identified sterol composition as quantified by GC–MS, (c) LDs formation and Nile Red fluorescent intensity, and the chromatograms of the change in (d) sterol composition and (e) TAG content of yeast cells after treatment with ECQ of S. cerevisiae wild-type and ∆erg6 strains treated with the Xylaria extract or ECQ. Gene labelled in green or red colour indicated up- or down-regulated expression, respectively. Asterisks indicated target of Morpholines. Red arrows indicated increased or decreased accumulation of sterol level, following the ECQ treatment. “ND.” Was referred to “not detected”. Error bars represent standard error of the mean (SEM). Different letters above the error bars (a–f) indicate significant differences at p value of < 0.05.

Figure 5

Mode of action of antifungal ECQ in the model yeast S. cerevisiae. (a) In wild-type strain, once entered the cells (1), ECQ not only disrupted actin cytoskeleton organization but also strongly induced expression of ERG genes, including ERG6, to compensate for lower ergosterol level (2). ECQ treatment resulted in the alteration of lipid composition (3), leading to increased accumulation of TAG as well as zymosterol and toxic sterol squalene that normally being stored in LDs. LDs formation was induced (4) while some aggregated actin and damaged protein were also removed via endosomes (5) to reduce effect of ECQ cytotoxicity. (b) In contrast to the ∆erg6 strain, membrane permeability was increased (1), allowing penetration of drugs including ECQ. Loss of Erg6 caused pronounced effects on ECQ-mediated inhibition of actin cytoskeleton organization and function (2) and alteration of ergosterol metabolism (3), resulting in elevated levels zymosterol, the defective plasma membrane and abnormal cell morphology. Additionally, despite formation of LDs, they were clustered and non-functional (4), resulting in increased accumulation of toxic squalene. However, newly formed LDs could not be properly destined for vacuolar degradation as a result of ECQ inhibition on actin transport of LDs (5). Overall, ECQ displayed a coupling antifungal mechanism, resulting in abnormal actin cytoskeleton organization and altered sterol and lipid homeostasis. Red arrows indicated a decreased or increased of cellular sterol or TAG levels after treatments with ECQ. Yellow star number 1–5 indicated proposed cellular events during ECQ treatments.