Landscape of essential growth and fluconazole-resistance genes in the human fungal pathogen Cryptococcus neoformans
Fig 4
Using TN-seq to assay genetic contribution to fluconazole resistance.
(A) TN-seq libraries were selected with IC50 levels of fluconazole dissolved in DMSO or with just the equivalent amount of DMSO as a control. Libraries were sequenced at time 0 and after two days growth in DMSO or fluconazole to identify genes with differential transposon insertion frequencies after selection. (B) Volcano plot of 5296 genes with 5 or more insert sites (out of 6,975 C. neoformans genes) displaying the mean log10 (Fluconazole/DMSO) value on the x-axis and the −log10 (Bonferroni corrected p-value) on the y-axis. Individual genes are shaded orange if the distribution of inserts is statistically different from the distribution of inserts into noncoding regions (p < 0.05 via Mann–Whitney U test after Bonferroni correction). Genes that are not statistically different are shaded blue. (C) Boxplot displaying distribution of log10-adjusted fold changes in insert density (i.e., frequency in fluconazole/frequency in DMSO). Boxplots show first quartile, median, third quartile. The whiskers show the range to a maximum of 1.5 times the interquartile range above and below the first and third quartile, respectively. Outlier data points (outside the whiskers) are not displayed. Not displaying outliers results in 15,902 of 454,341 intergenic sites, 1 of 107 sites from afr1, 1 of 4 sites from nap1, 1 of 34 sites from rim23, 2 of 127 sites from rra1, 12 of 74 sites from rim20, 0 of 5 sites from vps25, 5 of 77 sites from rim101, 5 of 151 sites from rim 13, and 1 of 11 sites from vps23 not being displayed although those data were considered in the statistical analyses. Snf7 is not shown because there were 0 inserts between the start and stop codons. Inserts in intergenic regions are indicated in grey, genes where inserts were significantly depleted after fluconazole treatment are shown in orange (afr1, rim23, rra1, rim20, rim101, rim13, vps23) and genes where inserts were not significantly depleted after fluconazole are shown in blue (nap1 and vps25). Notably, both nap1 and vps25 had very low numbers of inserts that limited statistical power. (D) Spot dilution assays with 5 μL spots plated. The initial leftmost spot is of OD600 = 20 culture and each successive spot is a 10-fold dilution, so that the final spot should be 105 less concentrated than the first. Both plates were spotted on the same day with the same dilution series. All plates were imaged after 48 h at 30°C. Mutants were spotted on two separate plates for YPD and fluconazole media, each with a wildtype H99 control present. Data underlying B can be found in S1 Table. Data underlying C can be found in S1 Data at 10.5281/zenodo.15264486. Original images in panel D can be found in the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-2480.