FIGURE 5.

Anti-Candida activity of tempol depends on expression of HAP43. (A) Δ/Δ hap43 knockout mutant is less susceptible to tempol. Fungal viability was assessed using ATP quantification in three biological replicates with five technical replicates each [n = 3(5)]. Fungal strains were grown overnight at 30°C and subcultured in SC medium for 4 h before being seeded in a 96-well plate containing RPMI medium with two different tempol concentrations (0.5 and 1 mg/ml) and incubated for 24 h. A Δ/Δ hap43 knockout mutant was compared to a revertant strain derived from the knockout mutant. (B) Δ/Δ sfu1 knockout mutant is more susceptible to tempol. Similar experimental setup as above in five biological replicates with four technical replicates each [n = 5(4)]. (C) Simplified scheme of iron homeostasis regulation in C. albicans adapted from Chen et al. (2011). (D) C. albicans is less susceptible to tempol under high iron than under low iron conditions. High and low iron conditions in YEPD were achieved as described in the section “Materials and Methods.” Pre-cultured cells were subsequently seeded in a 96-well plate containing either high or low iron medium with different tempol concentrations and incubated for 24 h. Fungal viability was assessed using ATP quantification in four biological replicates with four technical replicates each [n = 4(4)]. (E,F) HAP43 but not SFU1 promotes antifungal effect of tempol. Similarly as above we assessed viability of (E) Δhap43 and (F) Δsfu1 homozygous deletion mutants in comparison with respective revertant strains under high and low iron conditions in four biological replicates with four technical replicates each [n = 4(4)]. ATP was quantified after 24 h incubation with two different tempol concentrations (0.5 and 1 mg/ml). Scatter plots represent mean ± SD with ^*p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ns = not significant.