Figure 1.
Model of zinc-regulated gene expression.
A) Under high or normal zinc conditions Zap1 binds zinc and blocks the ZRE elements of the ZRT1 promoter and therefore inhibits GFP expression. B) Under zinc deficient conditions Zap1 releases zinc then disassociates from the ZRT1 promoter, which leads to the expression of GFP.
Figure 2.
Zinc responsiveness of ZRE-GFP promoter-reporter construct.
A) Comparison of Zn-dependent GFP fluorescence in untransformed yeast cells (gray bars) and yeast transformed with the ZRE-GFP promoter-reporter construct (black bars). Cells were grown in LZM medium with indicated zinc concentrations. GFP fluorescence and cell density were measured after 20 hours of incubation at 30°C. The inset shows a power law fit through the data. The excellent quality of the fit (the linear correlation coefficient between the fluorescence and the zinc concentration to the power −0.372 is R = 0.993) indicates that the fluorescence decreases with increasing zinc concentration in a scale-free manner. B) Metal specificity of the promoter-reporter construct. Transformed cells were grown in LZM either without (black bars) or with 100 µM (gray bars) of indicated metal ions and analyzed as before. GFP fluorescence is expressed as percent of non-supplemented control. The fluorescence signal was always normalized by cell density. Error bars represent standard deviation (SD).
Figure 3.
Dose and time response of GFP fluorescence after treatment with TPEN.
A) Cells transformed with the ZRE-GFP promoter-reporter construct were grown in RPMI-1640 medium with indicated TPEN concentrations for 20 hours at 30°C. Fluorescence signal was normalized by cell density and is expressed as x-fold increase of untreated sample. B) Cells containing the ZRE-GFP promoter-reporter construct were grown in absence or presence of 5 µM TPEN in RPMI-1640 medium at 30°C. Fluorescence and cell density were measured at times indicated. Fluorescence signal is expressed as x-fold increase of the 0 hour time point. Error bars represent standard deviation (SD).
Table 1.
Summary of robustness criteria of the promoter-reporter assay.
Figure 4.
Screen results are reproducible.
The GFP intensities of replica plate sets of screen 1 and screen 2 were plotted against each other. The linear regression between both plate sets demonstrates reproducibility of hit compounds. The cut off level for hit selection is 1.5-fold. The red box shows hits with high GFP induction level.
Figure 5.
3.9% of all screened compounds showed GFP induction at 100 µM concentration (group B). Compounds that proved cytotoxic at 100 µM (4%) were serially diluted and reanalyzed (group A). Positive hits of both groups were categorized into classes according to their degree of GFP induction. Category 1 hits showed induction of 80% and higher. For categories 2 and 3 GFP induction was 50%–80% and 30%–50%, respectively. The percentage and number of compounds (in brackets) are shown for each class and category.
Figure 6.
A subset of hit compounds was subjected to flow cytometry analysis. Cells were treated with hit compounds and incubated for 20 hours at 30°C. Labile zinc content was measured using the zinc fluorescent probe Zinbo-5. Zinbo-5 fluorescence was plotted in comparison to GFP fluorescence.
Figure 7.
Total and labile zinc and cell morphology of C. albicans.
Candida cells treated with A, B) Atovaquone, C, D) Halofantrine or E, F) Disulfiram and incubated at 30°C for 20 hours. 100 µl aliquots were immediately used for flow cytometry, the remaining culture was used for ICP-MS analysis. Light microscopy images were obtained from cells treated with 25 µM atavaquone and halofantrine and 1 µM disulfiram using standard protocols. Error bars represent standard deviation (SD). Level of significance are characterized as followed (* P<0.01, ** P<0.001, *** P<0.0001, not significant P>0.05). Statistical significance was tested by one-way ANOVA.
Figure 8.
The cell model shows the four potential mechanisms of action for hit compounds to interfere with zinc homeostasis. Hit compounds can inhibit zinc uptake transporter, induce the formation of chelators or act as chelators themselves, interfere with zinc transport in or out of compartments, or modulate zinc transcription factors like Zap1.