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Comparative Study
. 2006 Aug;50(8):2732-40.
doi: 10.1128/AAC.00289-06.

Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae

Veronika Simons  1 , John P Morrissey, Maita Latijnhouwers, Michael Csukai, Adam Cleaver, Carol Yarrow, Anne Osbourn
Affiliations
Comparative Study

Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae

Veronika Simons et al. Antimicrob Agents Chemother. 2006 Aug.

Abstract

Many plant species accumulate sterols and triterpenes as antimicrobial glycosides. These secondary metabolites (saponins) provide built-in chemical protection against pest and pathogen attack and can also influence induced defense responses. In addition, they have a variety of important pharmacological properties, including anticancer activity. The biological mechanisms underpinning the varied and diverse effects of saponins on microbes, plants, and animals are only poorly understood despite the ecological and pharmaceutical importance of this major class of plant secondary metabolites. Here we have exploited budding yeast (Saccharomyces cerevisiae) to investigate the effects of saponins on eukaryotic cells. The tomato steroidal glycoalkaloid alpha-tomatine has antifungal activity towards yeast, and this activity is associated with membrane permeabilization. Removal of a single sugar from the tetrasaccharide chain of alpha-tomatine results in a substantial reduction in antimicrobial activity. Surprisingly, the complete loss of sugars leads to enhanced antifungal activity. Experiments with alpha-tomatine and its aglycone tomatidine indicate that the mode of action of tomatidine towards yeast is distinct from that of alpha-tomatine and does not involve membrane permeabilization. Investigation of the effects of tomatidine on yeast by gene expression and sterol analysis indicate that tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells accumulate zymosterol rather than ergosterol, which is consistent with inhibition of the sterol C(24) methyltransferase Erg6p. However, erg6 and erg3 mutants (but not erg2 mutants) have enhanced resistance to tomatidine, suggesting a complex interaction of erg mutations, sterol content, and tomatidine resistance.

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Figures

FIG. 1.
FIG. 1.
Differential effects of α-tomatine and tomatidine on Saccharomyces cerevisiae. A. Structure of the tomato leaf saponin α-tomatine. The site of cleavage by fungal extracellular enzymes to yield the aglycone tomatidine is indicated. B. The sensitivities of wild-type S. cerevisiae strains INVSc1 and KT1115 to α-tomatine and tomatidine were measured in agar plate assays. The strains were pregrown in YEPD and the cell densities adjusted to 2 × 107 cells/ml. This cell suspension and 1:10, 1:100, and 1:1,000 dilutions were replica plated onto YEPD containing different concentrations of α-tomatine or tomatidine. Growth tests were carried out at a range of pH values, since the antifungal activity of α-tomatine is pH dependent. C. Electrolyte leakage measurements. Cells of S. cerevisiae strain INVSc1 were suspended in distilled water (5 × 108 cells/ml) and treated with α-tomatine, tomatidine, a solvent control (DMF), or a lysis control (chloroform). Conductivity, which is a measure of leakage of electrolytes from the cell (cell lysis), was measured over time. Mean values for three independent experiments are presented, with bars indicating standard error values.
FIG. 2.
FIG. 2.
Effects of fenpropimorph, tomatidine, and α-tomatine on expression of ergosterol biosynthetic genes. The S. cerevisiae ergosterol biosynthetic pathway is shown along with the genes catalyzing each step. The expression levels of each gene in response to treatment with fenpropimorph, tomatidine, or α-tomatine were measured and the change (n-fold) relative to control treatment determined. Each graph shows this change with the bars indicating 95% confidence intervals. Any line that does not intersect unity (1.0) represents a statistically significant change in expression. Acetyl-CoA, acetyl coenzyme A.
FIG. 3.
FIG. 3.
Northern blot analysis of effects of chemical treatments and genetic mutations on gene expression. S. cerevisiae strain S288C was grown in the presence of α-tomatine, tomatidine, or the fungicides flutriafol and fenpropimorph. Controls included untreated yeast cells and cells grown in the presence of the solvent used to solubilize the antifungal compounds (1% DMSO). The erg mutants LPY11 (erg6), LPY25 (erg3), LPY27 (erg2) and the parent strains KT1357 and KT1358 were grown without drug treatment. The X2180-1A upc2-1 mutant (UPC20) and corresponding wild-type strain X2180-1A were also included in these experiments. Strains were grown aerobically to mid-log phase in YEPD medium. Total RNA was extracted and analyzed by hybridization with probes specific for ERG3, ERG26, DAN1, TIR3, PAU1, and UPC2. The bottom panel indicates rRNA abundance as assessed by ethidium bromide staining.
FIG. 4.
FIG. 4.
Tomatidine inhibits sterol biosynythesis. Analysis of sterol content following treatment of yeast strain S288C with α-tomatine or tomatidine. Peaks: A, cholesterol (added as an internal standard); B, zymosterol; C, ergosterol; D, ergosta-5,7-dienol.
FIG. 5.
FIG. 5.
Sensitivity of yeast sterol mutants to α-tomatine and tomatidine. The S. cerevisiae ergosterol biosynthetic mutants erg2, erg6, and erg3, as well as parent strain KT1357, were grown on agar plates (YEPD, pH 6.5) containing the membrane pemeabilizing agent nystatin, α-tomatine, or tomatidine as previously described for Fig. 1. All three mutants showed enhanced resistance to nystatin whereas differential effects were observed for tomatidine and α-tomatine.
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