Figure 3.

Hrr25 is required for cell wall and membrane stress tolerance and enables target-mediated echinocandin resistance

(A) Transcriptional repression of HRR25 reduces caspofungin and fluconazole tolerance of C. albicans. Wild type (CaSS1) and tetO-HRR25/Δ strains were grown overnight in YPD and then subjected to an additional overnight culture in the absence and presence of 0.05 μg/mL DOX. Dose-response assays were performed in YPD with 2-fold dilution gradients of the antifungal, in the presence or absence of DOX (0.5 μg/mL) as indicated. Growth (OD₆₀₀) was measured after 72 h of incubation at 30°C, averaged between technical duplicates, and normalized to the wild-type drug-free control (see color bar). All assays were performed in biological duplicate. See also Figure S2.

(B) Transcriptional repression of HRR25 reduces echinocandin and azole susceptibility in C. albicans clinical isolates. DPL15 represents an echinocandin-resistant clinical isolate harboring a mutation in the drug target gene FKS1. CaCi-2 and CaCi-17 represent azole-tolerant and azole-resistant clinical isolates obtained from a patient undergoing azole therapy. Parental and HRR25 conditional-expression strains were grown overnight in YPD and then subject to an additional overnight culture in the absence and presence of DOX (0.5 μg/mL for DPL15 and CaCi-2 backgrounds; 5 μg/mL for CaCi-17 background to achieve sufficient transcriptional repression). Dose-response assays were performed as described in (a), in the presence or absence of DOX (0.5 μg/mL for DLP15 and CaCi-2 backgrounds; 5 μg/mL for CaCi-17 background) as indicated. Growth (OD₆₀₀) was measured after 72 h. All assays were performed in biological duplicate. See also Figure S2.

(C) Transcriptional repression of HRR25 enhances sensitivity to the cell wall-perturbing agent calcofluor white and the membrane-perturbing agent sodium dodecyl sulfate. Strains were cultured as described in (b) and subject to dose-response assays as described in (a). Growth was measured after 48 h for calcofluor white and 72 h for sodium dodecyl sulfate. All assays were performed in biological duplicate. See also Figure S2.

(D) Deletion of SWI4 and SWI6 confers hypersensitivity to cell wall and membrane stress. Wild type (CB420) and swi4Δ/Δ swi6Δ/Δ strains were grown overnight in YPD and dose-response assays were performed as described in (a). Growth was measured after 72 h for caspofungin and fluconazole and 48 h for calcofluor white and sodium dodecyl sulfate. All assays were performed in biological duplicate.