FIGURE 9: Clinically relevant concentrations of lithium chloride and ebselen significantly reduce capsule and/or biofilm formation by C. neoformans in vitro. (A) DIC microscopy images of C. neoformans H99S wild-type cells grown in CIM (control) or CIM supplemented with 1 mM lithium chloride for 48 h and stained with India ink to visualize capsule. Scale bar, 10 µm. (B) Quantification of cell diameter and capsule thickness for cells from panel A. The experiment was performed twice, and at least 100 cells were analyzed per strain and condition. Small grey squares and blue circles indicate individual data points from both experiments, and the black bar indicates the median ± interquartile range. ns, not significant. ****P < 0.0001 by t-test. (C) Brightfield microscopy images of C. neoformans H99S grown under biofilm-inducing conditions without (control) or with 20 µg ml^-1 ebselen (+ EBS) for 48 h. Scale bar, 20 µm. (D) Quantification of biofilms from panel C by the XTT reduction assay. OD₄₉₂, optical density at 492 nm. Results are the mean ± SEM of three independent experiments, each performed in triplicate. ****P < 0.0001 by t-test. (E) Proposed model of proteostasis-dependent capsule and biofilm inhibition by lithium. The PKA-pathway regulates proteasome homeostasis [33], which is required for robust capsule formation and biofilm production, both of which contribute to virulence. Lithium influences both PKA activity and proteostasis, resulting in reduced capsule and biofilm formation.

33. Geddes JM, Caza M, Croll D, Stoynov N, Foster LJ, Kronstad JW (2016). Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation. MBio 7(1): e01862-01815. https://doi.org/10.1128/mBio.01862-15