Cryptococcus neoformans Iron Homeostasis

The serotype A strain H99 of Cryptococcus neoformans var. grubii was used for all experiments and was maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). The Cfo1-GFP strain was constructed as described previously [9 (link)]. Growth under low iron conditions was performed in yeast nitrogen base (YNB) without amino acids media plus 2% dextrose at pH 7.0 with the addition of the iron chelator bathophenanthroline disulfonate (100–150μM BPS) (YNB-BPS). Defined low-iron media (LIM) was prepared as described [53 (link)] with the addition of 20 mM HEPES and 22mN NaHCO₂. Mammalian iron sources such as human hemoglobin (2μg mL^-1), bovine heme (10–100μM), human holo-transferrin (50μg mL^-1), and sheep blood (0.05%), as well as ferric chloride (FeCl₃) (10–100μM) were added to cultures. Different compounds were added at the following concentrations: 500nM N-Ethylmaleimide (NEM), 20μg mL^-1 brefeldin A (BFA), 500μg mL^-1 monensin, 150ng mL^-1 tunicamycin (TNC), 25μM aminodarone (AMD), 100μg mL^-1 cyclosporine A (CSA), 500ng mL^-1 tacrolimus (FK506), 5mM or 50mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 50μg mL^-1 rotenone, 1mM malonic acid, 10 mM salicylhydroxamic acid (SHAM). 2μg mL^-1 antimycin A, 5mM potassium cyanide (KCN), 0.01% hydrogen peroxide (H₂O₂), 50μM plumbagin, 5μg mL^-1 menadione, 1 mg mL^-1 calcofluor white (CFW), 0.01% sodium dodecyl sulfate (SDS), 0.5mg mL^-1 caffeine, 1.5M sodium chloride (NaCl), 10 μg mL^-1 caspofungin, 1.6mM quinacrine, 6mM chloroquine, 10μg mL^-1 fluconazole and 50ng mL^-1 miconazole. All chemicals were obtained from Sigma-Aldrich.