Fig 1.
Amoeba resistance varies between strains of C. neoformans.
A. A schematic overview of the amoeba resistance assay. A lawn of Cryptococcus is grown on V8 media for 60 hours. A. castellanii are added to the center of the lawn. A. castellanii consume the cells across the plate for a period of 12 or 18 days (based on amoeba activity). Cryptococcus strains able to resist amoeba have smaller areas of consumption when imaged. B. A. castellanii in co-culture with C. neoformans expressing RFP on solid V8 media. White arrows indicate examples of C. neoformans—amoeba interaction. C. Boxplots representing amoeba resistance phenotypes for a diverse set of C. neoformans strains after 18 days of amoeba co-culture. The x-axis displays the strains assayed. The y-axis represents the area the amoeba consumed. Smaller clearance areas indicate greater resistance to amoeba.
Table 1.
Genetically diverse C. neoformans strains surveyed for amoeba resistance.
Fig 2.
Phenotypic variation in amoeba resistance in mapping populations derived from both C. neoformans and C. deneoformans crosses.
A. Representative images of plates from amoeba resistance assays. On the plates, the area consumed by amoeba are highlighted in yellow. Parental strains are shown in the middle panels, and transgressive segregants are on the left and right. B. A histogram displaying amoeba resistance of segregants in the C. neoformans cross. The x-axis represents the amoeba clearance area. Phenotypes of the two parental strains are indicated in orange (Ftc555-1) and blue (Bt22). C. A histogram of the C. deneoformans F1 progeny amoeba phenotypes. Phenotypes of the two parental strains are indicated in orange (431) and blue (XL280).
Fig 3.
Amoeba resistance QTL for C. neoformans and C. deneoformans.
A. Manhattan plot representing the association between genotype and amoeba resistance in the C. neoformans mapping populations. The dotted line indicates the significance threshold by permutation. B. Distributions of segregant phenotypes associated with the QTL peak on chromosome 8 for C. neoformans. The x-axis represents allelic state at the QTL peak. This peak explains 62% of the amoeba resistance variation (R2). C. Manhattan plot for amoeba resistance in the C. deneoformans mapping population. This peak explains 62% of the amoeba resistance variation (R2). D. Segregant phenotypes by chromosome 7 genotype for C. deneoformans. E. A magnified view of the 95% confidence interval of the C. neoformans QTL for amoeba resistance. Barred lines at the top are the 95% confidence intervals for the C. deneoformans and C. neoformans amoeba resistance QTL. F. Gene diagrams for the region around the BZP4 gene for Ftc555-1 and Bt22. UTRs are shown in yellow.
Fig 4.
Disruption of BZP4 reduces amoeba resistance.
A. Models of the genomic regions surrounding BZP4 in Bt22 and Bt45. B. Amoeba resistance assay for two independent gene deletions of BZP4 in the Ftc555-1 background and a closely related strain of Bt22, Bt45. Bars are colored for comparison between closely related strains. The x-axis represents the strain tested. The y-axis represents the area amoeba consumed.
Fig 5.
Melanization and amoeba resistance share the same QTL.
A. Manhattan plot representing the association between genotype and melanization in the C. neoformans plot. The y-axis represents the strength of the association between genotype and light reflected (the degree of melanization). The x-axis represents the genomic location of the haploblocks used in the associations. The dotted line represents a significance threshold determined by a permutation test. Representative images of parents and segregants on L-DOPA media are also included. Each colony is from the randomized plates employed for QTL mapping. Images are brightened 30% to better display the difference in pigmentation. B. Segregant phenotypes at the maximum significance value of the QTL on chromosome 8. The x-axis represents the segregant allele at the maximum significance of the QTL. The y-axis represents the light reflected off of the colony when melanized. Parental strains are indicated in orange (Ftc555-1) and blue (Bt22). C. A magnified view of the chromosome 8 QTL peaks for amoeba resistance and melanization illustrating the QTL overlap. D. Plot comparing amoeba resistance and melanization phenotypes. The x-axis represents melanization and the y-axis represents amoeba resistance. Each dot represents a single segregant. Segregants are colored by their allele at the chromosome 8 QTL. E. Relationship between amoeba resistance and melanization in natural isolates. The linear regression line, and the coefficient of determination, are indicated for the linear model fit to the data with the strain H99 (outlier, top left) excluded.
Fig 6.
BZP4 expression differs significantly between genotypes.
A. Points representing the difference in transcript abundance between conditions in transcript per million (TPM) in segregants with each parental allele under the chromosome 8 QTL. The y-axis represents the total transcript counts. Boxplots are colored by parental allele. Significance is determined by ANOVA. “**” p < 0.005; “***” p < 0.0005. B. A Venn Diagram displaying the number of genes with increased and decreased expression for amoeba and control conditions based on the parental allele. Genes with decreased expression are colored in orange and those with increased expression are colored in blue. C. A hierarchically clustered heatmap of genes that strongly correlate with BZP4 expression and are differentially expressed based on the BZP4 allele in amoeba treated samples. Rows represent genes. Columns represent individual strains (parents and segregants). The colors of each cell represents z-scores. Colored rows at the bottom represent strains by their BZP4 genotype and their amoeba sensitivity (area of clearance, measured in cm2).
Fig 7.
Amoeba resistance and measures of virulence are uncorrelated.
A. Barplots representing the internal proliferation rate of parental strains and segregants. Boxplots are colored by the chromosome 8 allele. Orange boxes have the Ftc555-1 allele and blue have the Bt22 allele, darker colors indicate segregants. Dots represent individual measurements. Strains are oriented in rank order of amoeba resistance (highest to lowest resistance). Significance determined by ANOVA (p = 0.29 F = 1.28). B. Survival curves for animals infected with the parental strains and a group of F1 segregants. H99 is in black, Ftc555-1 is in orange, and Bt22 is in blue. Segregant curves are colored by parental allele under the chromosome 8 QTL, dark blue represents the Bt22 allele and darker orange represents the Ftc555-1 allele.