MCC/eisosome proteins Sur7, Pil1, and Lsp1 are important for resistance of Candida albicans to copper.
(A) C. albicans strains lacking the indicated MCC/eisosome genes were replica-plated onto minimal medium containing either 0 μM or 500 μM CuSO4 and incubated at 30°C for 48 hr. Only the sur7Δ and the pil1Δ lsp1Δ double deletion demonstrated strong sensitivity. (B) SUR7 overexpression partially rescued the copper sensitivity of the pil1Δ lsp1Δ strain. pil1Δ lsp1Δ strains containing zero, one, or two extra copies of the SUR7 gene were spotted onto minimal medium in the absence or presence of 1 mM CuSO4 and incubated for 48 hr at 30°C. (C) The highest concentration of copper at which the indicated strains could grow after incubation in synthetic medium in 96-well plates for 48 hr at 37°C with different concentrations of copper. The sur7Δ, pil1Δ lsp1Δ, and known copper sensitive mutant cup1Δ crp1Δ could only grow at lower concentrations of copper than the wild-type strain, indicating increased sensitivity. (D) Time course study of viability after exposure to copper. Cells were incubated in H2O or 0.5 μM CuSO4 at 37°C for the indicated time and then an aliquot was spread on YPD medium to determine viable colony forming units (CFU). All deletion mutants exhibited lower viability after incubation in CuSO4. Images in panel A are representative of three independent experiments, and those in panel B of two independent experiments, all performed on different days. The results in panel C represent averages of three independent experiments and those in panel D of four independent experiments, all performed on different days. Error bars indicate SD. ****, P<0.001. ***, P<0.01. **, P<0.01 by two-way ANOVA. Strains used were: WT, wild-type DIC185; sur7Δ (YJA11); sur7Δ comp. (YJA12); pil1Δ lsp1Δ (YHXW21-1); pil1Δ lsp1Δ comp (+ LSP1, YHXW23-1); seg1Δ (YLD182-9-10-2); slm2Δ (YHXW36-1); pkh1Δ pkh3Δ/PKH3 (YLD146-11-2-5); pst1Δ pst2Δ pst3Δ ycp4Δ (LLF060); nce102Δ (YHXW14); pun1Δ (YLD204-2); fmp45Δ (YHXW3), pil1Δ lsp1Δ + 1xSUR7 (YHXW45-1), pil1Δ lsp1Δ + 2xSUR7 (YHXW46-1), and cup1Δ crp1Δ (KC25).