Fk506

The A. fumigatus wild-type strain (akuB^KU80), azole-resistant clinical isolates (F14946 and F16216), echinocandin-resistant strain (EMFR-S678P), CN pathway deletion strains including CN catalytic subunit deletion (ΔcnaA), immunophilin FKBP12 and cyclophilin A deletion strains (Δfkbp12; ΔcypA) and various cnaA mutated strains were utilized for antifungal susceptibility assays. Strains were cultured on GMM (glucose minimal medium) agar for 5 days at 37 °C for collection of conidia for the assays. Broth dilution antifungal susceptibility testing was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines in RPMI-1640 medium with slight modifications. Conidia were harvested in 0.05% Tween 80 and counted to obtain a spore concentration of 10⁴/mL and subsequently diluted to obtain 5 × 10²/mL in each well (200 µL) of a 96-well plate. Effect of CN585 (Sigma-Aldrich, St. Louis, MO, USA) or FK506 (Astellas Pharma, Tokyo, Japan) on growth was tested at concentrations ranging from 0 to 10 µg/mL. Interpretation of results on growth inhibition was performed after 48 h of growth at 37 °C by microscopic observation. Bright-field photomicrographs were acquired on a Nikon Diaphot phase contrast inverted microscope equipped with a Canon T5i digital camera.

Milbemycin α25 was a generous gift from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). FK506 was kindly provided by Astellas Pharma Inc. (Tokyo, Japan). FLC was purchased from LKT Laboratories Inc. (St. Paul, MN). Clotrimazole (CLT), miconazole (MCZ), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), nigericin (NIG), monensin (MON), latrunculin A (LaA), cerulenin (CER), rhodamine 6G (R6G), beauvericin, and amphotericin B (AMB) were obtained from Sigma-Aldrich Japan Inc. (Tokyo, Japan). Enniatin B was purchased from Alexis Biochemicals (San Diego, CA). Cycloheximide (CHX) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). RC21v3 was our in-house-developed d-octapeptide Candia albicans Cdr1-specific inhibitor (23 (link)).

All fungal strains and plasmids used in this study are listed in Table S1A in the supplemental material. For spore production, Mucor strains were inoculated and maintained on yeast extract-peptone-glucose (YPG; 3 g/liter yeast extract, 10 g/liter peptone, 20 g/liter glucose, 2% agar, pH 4.5) or yeast extract-peptone dextrose (YPD; 10 g/liter yeast extract, 20 g/liter peptone, 20 g/liter glucose, 2% agar, pH 6.5) agar at 26°C or 30°C for 4 days. cnbRΔ mutants were maintained on YPD agar at 30°C. For in vitro Mucor-host interaction studies or in vivo survival studies, cnbRΔ mutants were grown overnight in liquid YPD at 30°C with shaking. For high-CO₂ conditions, the flasks were entirely filled with YPD broth, and the wild type (R7B) was inoculated on the bottom of the flasks. The flasks were sealed with Parafilm and were left at room temperature overnight without being disturbed. For testing with calcineurin inhibitors, Mucor strains were grown in liquid YPD or on YPD agar plates supplemented with FK506 (Astellas Pharma Inc.) (1 μg/ml) or CsA (LC Laboratories) (2 or 100 μg/ml) at 30°C for 2 to 5 days.