Altered responses of copper sensitive morphogenesis mutants and flippase mutants to membrane-targeted drugs.
The susceptibility of copper-sensitive mutants to (A) amphotericin B, (B) cinnamycin, (C) duramycin and (D) papuamide A was determined by halo assays in which 2.5 x 105 cells of the indicated strain were spread onto the surface of minimal medium plates, filter discs containing different amounts of the drugs were placed on the surface of each plate, and then the zone of growth inhibition was quantified. (E) Susceptibility of C. albicans flippase mutants to duramycin and copper. The filter disks on one plate contained 10 μl of 0, 2.5, 5, 10, or 20 mg/ml duramycin. The other plate contained 0, 75, 150, or 300 mM CuSO4. The zone of growth inhibition (halo) surrounding each filter disc was recorded after incubation for 48 hr at 30°C. Note that there were no significant differences in halo sizes between the copper-sensitive mutants and the wild type for amphotericin B which targets ergosterol, but significant differences were detected for cinnamycin and duramycin that target phosphatidylethanolamine (PE) and for papuamide A that targets phosphatidylserine (PS). Graphs represent averages of three independent experiments performed on different days. A representative set of halo assays is shown in the supporting information (S5 Fig). None of the mutants were significantly different from the wild type for amphotericin B, but all of the mutants were significantly different from the wild type for the other drugs by two-way ANOVA (P < 0.05 or lower). Strains in panels A-D were wild type control DIC185, sur7Δ (YJA11), pil1Δ lsp1Δ (YHXW21-1), rvs161Δ (YLD14-3), rvs167Δ (YLD16), and arp2Δ arp3Δ (CaEE27). Strains in Panel E were wild type control DIC185, and the flippase mutants included neo1Δ (C1_04630CΔ; YLD224-9), dnf1Δ (C5_00570WΔ, YLD219-2-9-1) and drs2Δ (C3_07230WΔ; YLD220-14-18-1).