A chemical genetic screen reveals a role for proteostasis in capsule and biofilm formation by Cryptococcus neoformans

Abstract

Pathogenic microorganisms employ specialized virulence factors to cause disease. Biofilm formation and the production of a polysaccharide capsule are two important virulence factors in Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis. Here, we show that the bipolar disorder drug lithium inhibits formation of both virulence factors by a mechanism involving dysregulation of the ubiquitin/proteasome system. By using a chemical genetics approach and bioinformatic analyses, we describe the cellular landscape affected by lithium treatment. We demonstrate that lithium affects many different pathways in C. neoformans, including the cAMP/protein kinase A, inositol biosynthesis, and ubiquitin/proteasome pathways. By analyzing mutants with defects in the ubiquitin/proteasome system, we uncover a role for proteostasis in both capsule and biofilm formation. Moreover, we demonstrate an additive influence of lithium and the proteasome inhibitor bortezomib in inhibiting capsule production, thus establishing a link between lithium activity and the proteasome system. Finally, we show that the lithium-mimetic drug ebselen potently blocks capsule and biofilm formation, and has additive activity with lithium or bortezomib. In summary, our results illuminate the impact of lithium on C. neoformans, and link dysregulation of the proteasome to capsule and biofilm inhibition in this important fungal pathogen.

Keywords: Cryptococcus neoformans; biofilm; capsule; lithium; proteostasis.

Figure 1. 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 pka1Δ 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 pka1Δ 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 pkr1Δ 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. *P < 0.05, and ****P < 0.0001 by two-way ANOVA.

Figure 2. FIGURE 2: Phosphoglucomutase does not appear to be a major target of lithium in C. neoformans.

(A) Growth curve analysis for C. neoformans H99S in YNB medium containing either 2% glucose or 2% galactose as sole carbon source at 30°C in the absence (control) or presence of lithium chloride (LiCl). Solid lines represent the means of results from two independent experiments, each performed in quadruplicate, and shaded areas represent the standard errors of the means. (B) Relative growth data from panel A. Results were plotted relative to the untreated controls. Note that cells grown in galactose were less sensitive to elevated LiCl concentrations compared to cells grown in glucose. Glu, glucose; Gal, galactose.

Figure 3. FIGURE 3: Chemical genetic analysis of the impact of lithium on C. neoformans.

(A) Screening results for the 2008 C. neoformans knockout (CNKO) library. Results are based on OD₆₀₀ measurements and are plotted as log₂ ratio of growth in YPD medium supplemented with 100 mM lithium chloride relative to growth in YPD medium only. Roman numerals refer to the 96-well plate numbering as provided by the Fungal Genetics Stock Center (http://www.fgsc.net . Mutants significantly affected by lithium were identified by calculating the mean log₂-ratio of growth for all mutants of an individual plate and applying a cutoff of ± 1.5-fold the standard deviation (SD). Circles represent individual mutants. Blue colored circles indicate mutants that had significantly reduced (negative log₂-ratios) or increased (positive log₂-ratios) tolerance to lithium. Grey circles indicate mutants that did not show altered growth in presence of lithium. (B) Screening results for the 2015 CNKO library. See panel A for details. (C) Screening results for the transcription factor (TF) mutant library. See panel A for details. Note that for this smaller library, the mean and ± 1.5-fold SD cutoffs were calculated for mutants of all four plates combined instead of for individual plates. The mean is indicated by a solid black line, and the dashed lines indicate the 1.5-fold SD. (D) Proportion of mutants identified from the screen with reduced or increased tolerance towards lithium.

Figure 4. FIGURE 4: A STRING analysis reveals that multiple cellular pathways and activities are regulated by lithium in C. neoformans, including the ubiquitin/proteasome system.

The corresponding proteins of the 292 mutants found to be hypersensitive or hypertolerant to lithium were subjected to a STRING analysis using default settings. Network edges are based on confidence with line thickness indicating the strength of data support. Network clusters are color-coded and labeled. Note that the network was built based on data from C. neoformans strain JEC21. In some cases, the corresponding gene names for C. neoformans strain H99 are indicated. HOG, high osmolarity glycerol; PKA, protein kinase A; RAS, rat sarcoma.

Figure 5. FIGURE 5: The ubiquitin/proteasome-associated mutants identified in the lithium screen have defects in proteins implicated in several stages of the ubiquitin/proteasome system.

The strains defective in proteins belonging to a specific functional category of the ubiquitin/proteasome system (for example E1, or DUB) are color-coded and indicated based on their strain designation at the Fungal Genetics Stock Center (http://www.fgsc.net see Table 1). Strains in bold are those that were found to be defective in capsule formation (see Figure 6). Ub, ubiquitin; Nedd8, neural precursor cell expressed, developmentally down-regulated 8; SUMO, small ubiquitin-related modifier; URM1, ubiquitin-related modifier 1; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; DUB, deubiquitinating enzyme.

Figure 6. FIGURE 6: The ubiquitin/proteasome system contributes to capsule and biofilm formation.

(A) DIC microscopy images of the indicated ubiquitin/proteasome-associated C. neoformans strains (see Table 1) grown in CIM for 48 h and stained with India ink to visualize capsule via dye exclusion. H99C, Wt control for strains from the 2008 CNKO collection. KN99α, Wt control for strains from the 2015 CNKO collection. Scale bar, 5 µm. (B) Quantification of cell diameter (grey squares) and capsule thickness (blue circles) for cells from panel A. The experiment was performed twice, and at least 100 cells were analyzed per strain and condition. The black bar indicates the median ± interquartile range. The dotted black lines indicate the median capsule sizes of the respective Wt controls. ns, not significant. **P < 0.01 by two-way ANOVA compared to the respective Wt. (C) Biofilm formation by ubiquitin/proteasome-associated mutants. Indicated strains were grown under biofilm-inducing conditions without (control) or with 50 mM LiCl and quantified 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 < 0.05, and **P < 0.01 by t-test compared to the respective Wt growth condition.

Figure 7. FIGURE 7: Combination of lithium with the proteasome inhibitor bortezomib blocks capsule production.

(A) DIC microscopy images of the indicated C. neoformans strains grown in CIM (control) or CIM supplemented with the indicated concentrations of LiCl and/or bortezomib for 48 h and stained with India ink to visualize capsule. Wt, H99S wild type; BTZ, bortezomib. Scale bar, 5 µm. (B) Quantification of cell diameter and capsule thickness for cells from panel A. The experiment was performed twice, and at least 25 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. The dotted black lines indicate the median capsule sizes of the Wt or pkr1Δ mutant grown under control conditions. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by t-test. (C) Relative capsule thickness data from panel B. Results were plotted relative to the untreated controls. Note that capsule formation by the pkr1Δ mutant is less affected by BTZ, but more strongly reduced by LiCl compared to the Wt.

Figure 8. FIGURE 8: The lithium-mimetic drug ebselen inhibits capsule formation, and its activity is potentiated by combination with lithium or bortezomib.

(A) DIC microscopy images of the C. neoformans H99S wild type and the pkr1Δ mutant grown in CIM (control) or CIM supplemented with the indicated concentrations of LiCl and/or ebselen for 48 h and stained with India ink to visualize capsule. EBS, ebselen. Scale bar, 5 µm. (B) Quantification of cell diameter and capsule thickness for cells from panel A. The experiment was performed twice, and at least 64 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. The dotted black lines indicate the median capsule sizes of the Wt or pkr1Δ mutant grown under control conditions. ***P < 0.001, and ****P < 0.0001 by t-test. (C) Relative capsule thickness data from panel B. Results were plotted relative to the untreated controls. Note that capsule formation by the pkr1Δ mutant is less affected by EBS but more strongly reduced by LiCl compared to the Wt. (D) DIC microscopy images of the C. neoformans H99S wild type and the pkr1Δ mutant grown in CIM (control) or CIM supplemented with the indicated concentrations of bortezomib and/or ebselen for 48 h and stained with India ink to visualize capsule. BTZ, bortezomib; EBS, ebselen. Scale bar, 5 µm. (E) Quantification of cell diameter and capsule thickness for cells from panel D. 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. The dotted black lines indicate the median capsule sizes of the Wt or pkr1Δ mutant grown under control conditions. ns, not significant. ****P < 0.0001 by t-test. (F) Relative capsule thickness data from panel E. Results were plotted relative to the untreated controls. Note that capsule formation by the pkr1Δ mutant is less affected by BTZ and EBS compared to the Wt.

Figure 9. 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 , 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.