Fig 1.
Chemical structures of auroramycin, auroramycin aglycon, vicenistatin, incednine, silvalactam, salinilactam, and amphotericin B.
Table 1.
Antifungal activity of auroramycin.
Fig 2.
Auroramycin but not its aglycon derivative inhibits the growth of Saccharomyces cerevisiae.
Yeast cells were exposed to auroramycin and its aglycon at the indicated concentrations in duplicates in a 96-well microplate. Cell growth was quantified by recording the absorbance at 600 nm after 24 hours. Growth (normalized with respect to DMSO-treated cells) is plotted against concentration of the compounds. Error bars (n = 2 Mean ± S.D) are indicated in the plots.
Fig 3.
Homozygous profiling of auroramycin in Saccharomyces cerevisiae.
(A)log FC (Fitness Coefficient) was plotted against P-value for the 4817 mutants that were analysed by HOP. Mutants with higher sensitivity to auroramycin have more negative log FC values. Individual mutants are indicated as blue circles. Mutants sensitive to auroramycin (log FC < -0.5 and P-value < 0.05) are indicated as red circles. For the sake of clarity, gene names of only a few of the top hits are shown in the plot. (B)Wild type BY4743 and 23 deletion strains that were sensitive to auroramycin in chemogenomic profiling assay, were grown in YPD-HEPES in the presence of either DMSO or auroramycin at 1.56 μM in duplicate. Normalized growth after 16 hours of incubation at 30 °C is plotted for each strain. Error bars (n = 2 Mean ± S.D) are indicated in the plots. Representative data from two independent experiments are shown in the figure.
Fig 4.
Gene Ontology (GO) analysis of genes conferring resistance to auroramycin.
(A)Enrichment of GO categories in Biological process, Cellular component and Molecular function among the top cluster of genes conferring resistance to auroramycin (log FC < -0.5 and P-value < 0.05) was determined using DAVID. Number of genes in the GO category is indicated by the horizontal bar. Details of genes in each category are provided in the S1 Table. (B)Enrichment of GO categories in Biological process among the top genes conferring resistance (log FC value < -0.5 and P-value < 0.05) to auroramycin was visualized using REVIGO. Bubble colour refer to the p-value for the false discovery rates whilst circle size gives the frequency of the GO term in the underlying GO database (bubbles of more general terms are larger; http://revigo.irb.hr/).
Fig 5.
Comparison of sensitivity of mutant strains to auroramycin and amphotericin B.
Growth of wild type strain BY4743 and 8 deletion strains in auroramycin or amphotericin B at the indicated concentrations in duplicate. Normalized growth after 24 hours of incubation at 30 °C is plotted for each strain. Error bars (n = 2 Mean ± S.D) are indicated in the plots.
Fig 6.
Auroramycin’s inhibitory effect is not via interaction with ergosterol.
BY4743 cells were incubated in YPD-HEPES medium the presence of indicated concentrations of auroramycin (top) or amphotericin B (bottom) premixed with different concentrations of ergosterol in duplicate in a 96-well plate. Average growth of the duplicate cultures is plotted against the concentration of either auroramycin (upper panel) or amphotericin B (lower panel). Plots indicating the duplicate OD values with a picture of the corresponding plate are presented in S4 Fig. Representative data of one of two independent experiments are shown here.
Fig 7.
Auroramycin disrupts the vacuolar structure of yeast cells.
Wild type yeast cells containing vacuoles pre-labelled with the lipophilic dye FM 4–64 were exposed to DMSO, auroramycin, auroramycin-aglycon or amphotericin B at the indicated concentrations for 1 hour. Vacuolar morphology was analysed by fluorescence microscopy. (A)Based on the FM 4–64 staining patterns, yeast cells were classified into three categories: Single vacuole or multiple vacuoles or defective vacuoles. Representative images for each category are depicted. (B)Quantification of the various categories for cells treated with auroramycin, auroramycin-aglycon or amphotericin B (n = 100 cells).
Fig 8.
Auroramycin disrupts the membrane permeability of yeast cells.
Fluorescence reading (emission at 525 nm, excitation at 495 nm) in the supernatants of CFDA-labelled wild type yeast cells at different time points, after treatment with DMSO, cycloheximide or various concentrations of auroramycin or amphotericin B in triplicates. Error bars (n = 3 Mean ± S.D) represent standard deviation. Representative data from two independent experiments are shown.
Fig 9.
KCl but not sorbitol suppresses the inhibitory effect of auroramycin.
Wild type yeast strain (BY4743) was grown in YPD medium containing different concentrations of auroramycin and either KCl or sorbitol at the indicated concentrations in duplicate in a 96-well plate. Average growth of the duplicate cultures at the different concentrations after 16 hours of incubation at 30 °C is plotted. Plots indicating the duplicate OD600 nm values with a picture of the corresponding plate are presented in S5 Fig. Representative data from two independent experiments are shown.
Fig 10.
Auroramycin causes hyperpolarization of yeast cells.
(A)diS-C3(3) fluorescence of wild type AH109 yeast cells suspensions at different concentrations of amiodarone. (B)diS-C3(3) fluorescence of wild type AH109 yeast cells suspensions at different concentrations of auroramycin. (C)diS-C3(3) fluorescence of wild type AH109 yeast cells suspensions at different concentrations of auroramycin-aglycon. (D)Ratio of fluorescence at 585 nm to 575 nm is plotted for various cultures treated in A-C. Error bars (n = 3 Mean ± S.D) are indicated in the plot. Asterisks indicate statistical significance of fluorescence ratios for amiodarone/auroramycin-treated cells versus DMSO-treated cells, as determined by the Student’s t test (**, 2-sided P < 0.01 and *, 2-sided P< 0.05). This experiment was performed twice and data from one experiment are shown here.
Fig 11.
Auroramycin enhances the toxicity of positively charged drug hygromycin.
(A)Heatmap for fitness (growth percent) of the dose interaction matrix. (B)Corresponding heatmap for the synergy calculations, based on bliss independence model. Synergy is calculated based on bliss calculations less growth percent. (C)Representative growth rates at 8.9 μM hygromycin and 1.0 μM auroramycin (labelled as (1) in A, B), in comparison to the bliss independence model calculation, P < 0.003. Experiments were performed in triplicates. Error bars (n = 3 Mean ± S.D) are indicated in the plot.