Copper permeabilizes plasma membranes of C. albicans copper-sensitive strains with morphogenesis defects.
(A) Log phase sur7Δ cells were incubated in water containing the indicated concentrations of CuSO4 for 2 hr at 37°C and then stained with SYTOX Green to assess the integrity of the plasma membrane. The sur7Δ mutant displayed increased permeabilization of the plasma membrane with increased copper concentration compared to the wild type and Sur7 complemented strains. (B) Time course assay in which log phase sur7Δ cells were incubated in 10 μM CuSO4 for the indicated time at 37°C and then stained with SYTOX Green. The sur7Δ cells suffered greater membrane damage as early as 30 min after addition of copper.(C) Log phase cells of the morphogenesis mutants rvs161Δ, rvs167Δ, and arp2Δ arp3Δ strains were incubated in 10 μM CuSO4 for 2 hr at 37°C and then stained with SYTOX Green. All mutants demonstrated increased staining, indicative of membrane permeabilization. (D) Log phase cells of known copper mutants were incubated in 10 μM CuSO4 for 2 hr at 37°C and then stained with SYTOX Green. The crp1Δ, crp1Δ cup1Δ, and cup2Δ strains all showed significantly less staining than the sur7Δ strain, indicating a low level of membrane permeabilization caused by CuSO4. Error bars indicate SD. ****, P<0.001. ***, P<0.01. **, P<0.01, *, P<0.05 by two-way ANOVA in comparison to the corresponding wild type values. The results represent the average of three independent assays (panels A, C, D) or six independent assays (Panel B) carried out on different days. Strains used included DIC185, sur7Δ (YJA11), crp1Δ (KC16), cup1Δ crp1Δ (KC25), and cup2Δ (YLD117-1).