Cultivation of Cryptococcus Titan Cells

Strains used in this study are summarized in S1 Table. C. neoformans H99 [58 (link)], gpa1Δ, cac1Δ[34 (link)], and pka1Δ[59 (link)] were gifts from Andrew Alspaugh, Duke University, NC, USA. The gpr4Δ gpr5Δ [60 (link)] was kindly provided by Joseph Heitman, Duke University, NC, USA. Strains ric8Δ and usv101Δ were obtained from the Madhani 2015 collection (NIH funding, R01AI100272) from the Fungal Genetics Stock Centre and were validated by PCR and shown to phenotypically match published strains [36 (link), 37 (link)]. C. gattii R265 [61 (link)] was provided by Neil Gow, University of Aberdeen, UK. Isolates are summarized in S1 Table. Cells were routinely cultured on YPD (1% yeast extract, 2% bacto-peptone, 2% glucose, 2% bacto-agar) plates stored at 4°C. For routine culture, cells were incubated overnight in 5 mL YPD at 30°C, 150 rpm. For Titan induction, cells were incubated overnight at 30°C, 150 rpm in 5 mL YNB without amino acids (Sigma Y1250) prepared according to the manufacturer’s instructions plus 2% glucose. Fetal Calf Serum (FCS) was obtained from either BioSera (Ringmer, UK) or Sigma, which both induced Titan cells to a similar degree. FCS was routinely stored in 5 ml aliquots at -20 to prevent repeated freeze-thaw cycles. FCS was heat-inactivated by incubation at 56°C for 30 min.