FIG 3.

Ada2 controls antifungal drug tolerance and cell wall integrity. (A) The ada2 mutants were susceptible to azoles, echinocandins, and amphotericin B. Cells were grown overnight in liquid YPD at 37°C, washed twice with dH₂O, 5-fold serially diluted, and spotted onto solid YPD containing fluconazole (FLC), posaconazole (PSC), voriconazole (VRC), micafungin (MCF), caspofungin (CSF), or anidulafungin (ANF) at the indicated concentration. To test susceptibility to amphotericin B (AmB), strains were spotted onto synthetic complete (SC) medium with or without 50 ng/ml AmB. All plates were incubated at 37°C for 30 h. (B) A disk diffusion assay was used to test the susceptibility of the C. glabrata strains to FLC, MCF, or AmB. C. krusei ATCC 6258 and C. parapsilosis ATCC 20019 served as quality control strains. Cells were spread on the surface of Mueller-Hinton agar plus GMB medium plates. Disks containing 20 μg FLC, 40 ng MCF, or 2.5 μg AmB were placed on the agar surface, and the plates were incubated at 35°C for 24 h. (C) The ada2 mutants are sensitive to cell wall-perturbing agents but resistant to the ER stress chemical tunicamycin. Cells were grown overnight in liquid YPD at 37°C, 5-fold serially diluted, spotted onto solid YPD containing SDS, calcofluor white (CFW), Congo red (CR), dithiothreitol (DTT), or tunicamycin (TM), and incubated at 37°C for 30 h.