Fig. 4: Phosphatases involved in major virulence traits of C. neoformans.

a Growth curves of WT and phosphatase mutants were generated at 30 °C (control) and 37 °C (mammalian body temperature). Fifteen phosphatase mutants had growth defects at 30 and 37 °C. Nine of these phosphatase mutants had more substantial growth defects at 37 °C than 30 °C (additional data in Supplementary Fig. 8). Each curve represents data from two independent experiments (see Supplementary Fig. 8 for data from the individual experiments). Optical density at 600 nm (OD_600nm) was measured with a multi-channel bioreactor (Biosan Laboratories, Inc., Warren, MI, USA) for 40–90 h based on growth rate. b Melanin production was measured using three different melanin-inducing media (Niger seed, dopamine, and epinephrine medium). Representative images from three independent experiments are shown here. Each strain was spotted on medium containing 0.1% glucose, incubated at 30 °C, and photographed after 1–3 days. c, d Gene expression of the melanin-regulating genes LAC1, BZP4, and HOB1 were determined by qRT-PCR in both nutrient-rich (R) and nutrient-starvation (S) conditions. RNA was extracted from three biological replicates with three technical replicates of WT and melanin-regulating phosphatase mutants. Expression was normalized to ACT1, and statistical significance was calculated by one-way ANOVA analysis with Bonferroni’s multiple comparison test. Data are presented as mean values ± SEM (*P < 0.05, **P < 0.001, ***P < 0.0001). e The capsule production assay was performed using capsule-inducing media (FBS agar medium). Capsule thickness (total diameter−cell body diameter) was measured for WT cells (n = 50) and for each phosphatase mutant (n = 50). Statistical significance was calculated by one-way ANOVA analysis with Bonferroni’s multiple comparison test. Data are presented as mean values ± SEM (*P < 0.05, **P < 0.001, ***P < 0.0001). The graph is representative of more than three independent experiments. The images are representative DIC images of WT and phosphatase mutants incubated on FBS agar medium and stained with India ink. Scale bars, 10 µm.