Fig 1.
Conservation analysis of the C. neoformans STRIPAK complex.
(A) Table listing STRIPAK complex subunits used in BLAST analysis to identify protein orthologs in C. neoformans. (B) Sequence alignment of STRIPAK protein orthologs in C. neoformans, S. cerevisiae, and H. sapiens. Conserved protein domains are labeled in the schematic diagram: HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, TOR1), FHA (forkhead-associated domain), TM (transmembrane domain), and Mob1 (monopolar spindle-one-binder protein). (C) The Cryptococcus STRIPAK complex superimposed on the electron density map and ribbon model of the human STRIPAK complex using the program ChimeraX. The human complex is shown in grey, and the corresponding C. neoformans proteins are labeled in color. (D) AlphaFold structure prediction of CnSTRIPAK, with dotted lines representing pseudobonds.
Fig 2.
Yeast two-hybrid analysis of STRIPAK complex subunit interactions.
(A) S. cerevisiae cells carrying plasmids encoding the Gal4 DNA-binding domain (GBD) or the Gal4 transcriptional activation domain (GAD), fused to individual STRIPAK subunits, were crossed. The resulting diploid cells were grown on synthetic dextrose medium with or without histidine. Interaction between bait and prey proteins activates Gal4, driving the expression of ADE2 and HIS3, making cells less red (+histidine plates) and allowing growth on medium deficient in both histidine and adenine (-histidine plates). Results are representative of two independent experiments. (B) Illustration of positive protein-protein interactions detected in the yeast two-hybrid assay.
Fig 3.
Genome instability in STRIPAK complex mutants.
(A) Read depth analyses from whole-genome sequencing of the WT/WT (CnLC6683) parental diploid strain and heterozygous mutant diploid strains. The WT/WT genome is euploid, while PPH22/pph22Δ and MOB3/mob3Δ strains exhibit multiple instances of segmental and whole chromosome aneuploidy, with dark blue and orange highlighting regions/chromosomes with increased and decreased read depth, respectively. (B) Chromosome maps of pph22Δ and mob3Δ strains show aneuploidy in some, but not all, haploid progeny. Progeny can also acquire segmental or chromosomal aneuploidies absent in the parental strain. “P” stands for meiotic progeny from the indicated parent. (C) Coverage of far8Δ mutants generated in the H99 background reveals duplication of most chromosomes, suggesting a diploid state. Complementation of the deletion allele in the far8Δ::FAR8 strain restores euploidy or results in haploidy. Regions of read coverage in the genome maps in (A-C) are shaded based on Z >1.96, P <0.05 (dark blue); or Z < -1.96, P <0.05 (orange). (D) FACS analysis of pph22Δ and far8Δ strains. Cells were stained with propidium iodide to measure DNA content via flow cytometry. Peaks represent relative DNA content during G1/S and G2/mitotic phases. H99 and WT/WT (CnLC6683) (KN99a/KN99α) served as 1N and 2N controls, respectively. The number sign (#) after a strain name indicates F1 meiotic progeny dissected from a heterozygous mutant diploid parental strain. Graphs are representative of two biological replicates.
Fig 4.
Deletion of PPH22 in a diploid background leads to haploinsufficiency.
(A) Self-filamentation of WT/WT (CnLC6683) and PPH22/pph22Δ diploid strains on MS medium. Strains were grown on MS and incubated at room temperature (24°C) in the dark for 3 to 4 weeks before images were taken. Each spot represents an independent experiment. A representative image from one out of four independent experiments for each strain is shown. (B) Scanning electron microscopy (SEM) analysis of basidia and basidiospores from WT/WT and PPH22/pph22Δ strains. Samples were prepared following incubation on MS media for four weeks [shown in (A)]. The black arrowhead in image PPH22/pph22Δ-4 shows a hyphal filament being produced from a basidia head. Scale bars represent 5 μm. (C) Differential interference contrast (DIC) microscopy images of WT/WT and PPH22/pph22Δ cells grown to mid-logarithmic phase in YPD liquid media at 30°C. PPH22/pph22Δ cells showed elongated cells, increased cell size, incomplete budding, and abnormal cell clusters. Cultures were grown in biological replicates for analysis. Scale bars represent 10 μm. (D) Growth rates of WT/WT and PPH22/pph22Δ strains grown in YPD liquid culture at 30°C. Cell density was quantified as cells/mL, and logarithmic growth was modeled using nonlinear regression. (E) Competition assay of WT/WT versus PPH22/pph22Δ. The growth of strains in the coculture is expressed as a percentage of the total number of cells at the indicated time point. The data correspond to the mean of four biological replicates ± standard deviation.
Fig 5.
pph22Δ mutants frequently accumulate suppressor mutations.
(A) Representative image showing the growth of spores dissected from the PPH22/pph22Δ-4 strain on YPD medium. Wild-type spores are marked with a square, and pph22Δ deletion mutant spores with a triangle. The image was taken after five days of incubation at 30°C. (B) pph22Δ strains exhibit very slow growth and form tan-colored colonies. After prolonged incubation, large white colonies appear (indicated by black arrowheads), resulting from suppressor mutations. The image shows a YPD plate incubated at 30°C for one week. (C) Wild-type (KN99), isogenic pph22Δ, and pph22Δ suppressor (pph22Δ sup) strains were serially diluted and spotted onto YPD medium at 24°C, 30°C, and 33°C. Images were taken after 5 days of incubation. (D) pph22Δ sup strains show shared aneuploidy on chromosome 6. The diagram displays read coverage from whole-genome sequencing, with chromosomal coordinates at the bottom. The region of increased coverage shared by all 10 pph22Δ sup strains is indicated by a red box, which spans 7 genes. The read depth in this region is at least 3 times higher than the average depth of the rest of chromosome 6. (E) Fold change in expression of the 7 genes with a complete ORF included in the ~15 kb boxed region from (D), determined from RNA-seq. Fold changes in gene expression in pph22Δ (PP53, PP55, PP56) and pph22Δ sup (PP80, PP83, PP84) samples relative to wild type (WT, KN99a), were calculated by normalizing each gene’s FKPM to the housekeeping gene ACT1. Error bars represent the standard deviations calculated from biological triplicates. The gene ID numbers, from left to right in the graph, are CNAG_02234, CNAG_02235, CNAG_02236, CNAG_02237, CNAG_02238, CNAG_02239, and CNAG_02240. (F) AlphaFold structure prediction of Pph22 and Ppg1 and a superimposition of the two proteins. Root mean square deviation (RMSD) values of superimposed structures are 0.507 Å (between 285 pruned atom pairs) and 1.644 Å (across all 302 atom pairs).
Fig 6.
Deletion mutations in PPH22, FAR8, and MOB3 lead to defects in sexual differentiation.
(A) PPH22/pph22Δ and MOB3/mob3Δ strains exhibit defects in self-filamentation compared to WT/WT (CnLC6683). Images were taken after four weeks of incubation on MS plates at room temperature. Scale bars represent 200 μm. (B) Mating efficiency of STRIPAK complex mutants. pph22Δ, pph22Δ sup, far8Δ, mob3Δ, and far8Δ::FAR8 cells were co-cultured with isogenic wild-type cells of the opposite mating type (H99α or KN99a) on MS plates. An H99α x KN99a cross served as a control. Black arrows indicate basidia that did not produce spores in KN99a x α pph22Δ and KN99a x α far8Δ crosses. Scale bars represent 100 μm and 50 μm in the middle and bottom panels, respectively.
Fig 7.
Phenotypic analyses of pph22Δ and far8Δ mutants.
(A) WT (KN99a) and isogenic pph22Δ and pph22Δ sup strains were serially diluted and plated on YPD, YPD+1 M sorbitol, YPD+100 ng/mL rapamycin, YPD+1 μg/mL FK506, and YPD+100 μg/mL cyclosporine A (CsA) at 30°C, as well as YPD at 37°C. Images were taken between 2 and 5 days of incubation. This set is from the same experiment as Fig 5C. (B) WT (H99) and isogenic far8Δ-1, far8Δ-2, and far8Δ::FAR8, along with WT (YL99a) and isogenic far8Δ-3 and far8Δ-4 strains, were serially diluted and plated on the indicated media and temperature conditions. (C) WT, pph22Δ, pph22Δ sup, far8Δ, and far8Δ::FAR8 cells were serially diluted and spotted onto YPD supplemented with 3 mg/mL calcofluor white (CFW) or 0.5 mg/mL caffeine. Plates were incubated at 30°C and images were taken after 3 days. (D) DIC microscopy images depicting cell morphology of WT (H99), pph22Δ, and far8Δ strains grown to mid-logarithmic phase in synthetic complete (SC) media at 30°C. Scale bars represent 10 μm. (E) Fluconazole Etest to analyze drug susceptibility in WT (H99), far8Δ, pph22Δ, and pph22Δ sup strains. Cells were grown in an overnight culture in YPD to saturation, and then spread onto YPD plates before adding FLC Etest strips. Plates were incubated at 30°C and images were taken after 48 hours. Drug sensitivities are representative of two biological replicates.
Fig 8.
Role of PPH22 and FAR8 in production of virulence factors and pathogenicity.
(A) WT (KN99a, H99α, or YL99a) and isogenic pph22Δ, pph22Δ sup, far8Δ, and far8Δ::FAR8 strains were serially diluted and plated onto Niger seed agar medium to induce melanin production. The lac1Δ mutant was included as a negative control. Plates were incubated at 30°C for 7 days in the top panel and 4 days in the bottom panel. (B) Analysis of copper-dependent melanin production in pph22Δ and far8Δ mutants. The indicated strains were grown in a YPD overnight culture to saturation and spotted onto L-DOPA plates supplemented with either 10 μM of a copper chelator, bathocuproine disulfonate (BCS), or 10 μM copper(II) sulfate (Cu2SO4). The cbi1Δ ctr4Δ double mutant, which can only produce melanin in the copper-supplemented condition, served as a positive control. Plates were incubated at 30°C for 2 days. (C) Analysis of capsule formation by India ink staining of WT (KN99a or H99α), pph22Δ, pph22Δ sup, far8Δ, and far8Δ::FAR8 cells. Strains were grown for 3 days at 30°C in RPMI media to induce capsule formation. Cells were harvested, resuspended in PBS, and stained with India ink. The scale bar for the top panel of DIC images is 10 μm, and the scale bar for the bottom panel of images is 5 μm. (D) Cell size analysis of WT (H99), WT/WT (CnLC6683) (KN99a/KN99α), far8Δ, and far8Δ::FAR8 complemented strains. Cells analyzed were from the same experiment as (C). Images were analyzed with ImageJ/Fiji. Data are presented as a scatter dot plot with the indicated mean cell diameter. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (****, P <0.0001; ns, not significant) (WT, n = 190; WT/WT, n = 104; far8Δ-1, n = 151; far8Δ-2, n = 162; far8Δ::FAR8, n = 140). (E, F) Virulence of WT (KN99α), pph22Δ, pph22Δ sup, far8Δ, and far8Δ::FAR8 cells in a murine model of C. neoformans infection via the intranasal inhalation route. Equal numbers of male and female A/J mice (n = 14 per group) were inoculated with 105 cells and monitored for 45 days post-infection. (E) Survival rates of mice infected with the indicated strains. Ten mice per strain were analyzed. Dashed line indicates the time of fungal burden analysis (****, P <0.0001). (F) Brain and lungs were harvested from four randomly selected mice per group at 14 days post-infection to quantify CFU per gram of organ tissue (One-way ANOVA; ****, P <0.0001; ***, P <0.001; **, P <0.01; ns, not significant).
Fig 9.
mob3Δ mutants are hypervirulent.
(A) Thermotolerance of WT (KN99a) and isogenic mob3Δ strains. Cells were serially diluted, spotted onto YPD at 30°C, 37°C, 39°C, and 37°C with 5% CO2, and incubated for two days. (B) WT (KN99a) and mob3Δ cells were grown in RPMI at 37°C for three days, harvested, washed with PBS, and stained with India ink to analyze capsule formation. (C) Capsule thickness measurements were made by subtracting the cell body diameter from the capsule diameter and dividing by 2. Data are presented in a scatter dot plot with the mean capsule thickness indicated for each strain. Statistical analysis was performed using the Mann-Whitney U test (****, P <0.0001) (WT, n = 214; mob3Δ, n = 222). (D) Melanization of WT (KN99a) and mob3Δ strains on L-DOPA medium at 30°C after 24 and 48 hours of incubation. (E-H) Equal numbers of male and female A/J mice were infected intranasally with 105 cells of the indicated WT and isogenic mob3Δ mutant strains and analyzed for survival rate (n = 10) and fungal burden (n = 4). (E) Survival analysis of WT (KN99α) and mob3Δ #16 infected mice. Mice were 4 weeks old at the time of infection. The dashed line indicates the day at which fungal burden analysis was performed (***, P <0.001). (F) CFUs per gram of lung and brain tissue recovered from organs harvested at 14 days post-infection (Mann-Whitney U test; *, P = 0.014). (G) Survival rates of 5-week-old mice infected with WT (KN99α), mob3Δ #1, and mob3Δ #8 strains. The dashed line represents the day post-infection of fungal burden analysis (***, P <0.001). (H) CFUs per gram of lung and brain tissue recovered from organs harvested at 7 days post-infection (One-way ANOVA; **, P <0.01; *, P <0.05).
Fig 10.
Transcriptome analysis of pph22Δ, far8Δ, and mob3Δ mutants.
(A) Venn diagrams depicting shared differentially expressed genes (DEGs) among the indicated mutant strains for genes that were upregulated (left) or downregulated (right) compared to the isogenic wild-type control. Criteria for significant DEGs was log2 fold change >1.5 or <-1.5 and adjusted P value <0.05. (B, C, D) Volcano plots of -log10 adjusted P value versus log2 fold change for all DEGs in pph22Δ (B), far8Δ (C), and mob3Δ (D) mutants. Significant genes are highlighted as downregulated (blue) or upregulated (red). The top 10 most significantly regulated genes are labeled in each panel. The vertical and horizontal lines denote the log2 fold change thresholds (-1.5 and 1.5) and -log10(adjusted P value) threshold (2.0), respectively. (E, F, G) Heatmaps of hierarchical clustering depicting normalized expression data for selected virulence genes in pph22Δ, pΔ (E), far8Δ, fΔ (F), mob3Δ, mΔ (G), and wild-type, W samples. Row Z scores are color coded according to the legend at the right. Transcriptomic data for each mutant was gathered in biological triplicate and plotted with an independent set of wild-type control samples. (H, I, J) Gene ontology (GO) enrichment analysis of GO terms overrepresented in the transcriptomes of pph22Δ (H), far8Δ (I), and mob3Δ (J) samples. The top 15 significant GO terms are ranked based on the log2 fold enrichment score compared to the background set (all genes in the genome) and color coded by enrichment false discovery rate (FDR) to show significance [-log10(adjusted P value)].