FIG 7.

C. neoformans Cda1 catalytic point mutant strains fail to produce chitosan when grown under host-mimicking conditions. (A) Yeast strains were grown in YPD at 30°C for 72 h. Cells were collected, washed, and used for the measurement of cell wall chitosan by the MBTH assay. (B) The indicated strains were initially grown in YPD for 36 h, then collected, washed, and inoculated at a cell density of 500 cells/μl in RPMI medium containing 10% FBS, and incubated for 5 days at 37°C and 5% CO₂. At the end of incubation, cells were collected, washed, and used for chitosan measurement by the MBTH assay. Data are the averages for three biological experiments with each experiment having two technical replicates. Significant differences between the groups were compared by one-way ANOVA, followed by Bonferroni’s multiple-comparison test (****, P < 0.0001 comparing chitosan levels of strain KN99 to each other strain).