FIGURE 1: Lithium inhibits capsule and biofilm formation. (A) Dose-response growth assay for the indicated strains in YPD medium supplemented with increasing concentrations of lithium chloride (LiCl). The strains were incubated for 72 h at 30°C and the optical density was measured at 600 nm (OD₆₀₀). Note that the capsule-deficient pka1D mutant is significantly more sensitive to LiCl at concentrations ranging from 50 – 200 mM. Wt, wild type (H99S). Results are the mean ± SEM of two independent experiments, each performed in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 when comparing pka1D with the Wt by two-way ANOVA. (B) DIC microscopy images of the indicated C. neoformans strains grown in CIM (control), CIM + 100 mM NaCl, or CIM + 100 mM lithium chloride for 48 h and stained with India ink to visualize capsule via dye exclusion. Note that lithium strongly inhibits capsule formation in both the H99S Wt and the hypercapsular pkr1D mutant. Wt, wild type; NaCl, sodium chloride; LiCl, lithium chloride. Scale bar, 5 µm. (C) Quantification of cell diameter and capsule thickness for cells from panel B. The experiment was performed twice, and at least 100 cells were analyzed per strain and condition. Grey squares and blue circles indicate individual data points from both experiments, and the black bar indicates the median ± interquartile range. Ctrl, control. ns, not significant. ****P < 0.0001 by two-way ANOVA. (D) Brightfield microscopy images of the indicated C. neoformans strains grown under biofilm-inducing conditions without (control) or with LiCl for 48 h. Scale bar, 20 µm. (E) Quantification of biofilms from panel D by XTT reduction assay. OD₄₉₂, optical density at 492 nm. Results are the mean ± SEM of two independent experiments, each performed in sextuplicate. * < 0.05, and ****P < 0.0001 by two-way ANOVA.