The C. neoformans laboratory strain H99 served as the wild-type strain in this study. Stock cultures were preserved in a glycerol solution and stored at a temperature of −80°C. Cells were routinely cultured in a nutrient-rich medium called Yeast extract-Peptone-Dextrose (YPD) at a temperature of 30°C in a shaking incubator set to a moderate speed of 150–200 rpm. To solidify the medium, agar was added at a concentration of 2% (w/v). The drugs used in this study were dissolved in a solvent called dimethyl sulfoxide (DMSO) and stored at a temperature of −20°C.

H99 was thawed from the −80°C freezer and streaked onto YPD-agar plates, which were then incubated at 30°C for 72 h. After incubation, cells were resuspended in YPD broth and their densities were adjusted to 2.5 × 10³ cells/mL in YPD broth with or without TUN in a 96-well plate. The TUN concentrations were 0.03–0.125 μg/mL. The plate was then incubated at 30°C, and the optical density at 595 nm (OD₅₉₅) was monitored using a Tecan plate reader (Infinite F200 PRO, Tecan, Switzerland) at 15-min intervals for 72 h. The data are presented as the mean ± standard deviation (SD) of three biological replicates.

Cells were re-suspended in distilled water and adjusted to a concentration of 1 × 10⁷ cells/mL. Following this, 3 μL of 10-fold serial dilutions were spotted onto YPD agar plates with or without the presence of drugs. The petri dish plates were then incubated at 30°C and photographed after 72 h.

The broth microdilution method was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) M27-2017 guidelines (CLSI, 2017) with minor modifications.

Stock solutions of the drugs (TUN and FLC in YPD broth; 5FC in SD broth) were prepared and subjected to a twofold dilution process. A volume of 0.1 mL of each drug at varying concentrations was sequentially dispensed into U-shaped wells of 96-well microdilution plates (Thermo Fisher Scientific), resulting in final drug concentrations ranging from 0.125 to 128 μg/mL (FLC and 5FC) or 0.03–32 μg/mL (TUN).

To prepare the inoculum of test strains, 3–5 colonies were suspended in sterile saline and standardized to a turbidity of 0.5 McFarland using a spectrophotometer (NanoPhotometer® NP80, Implen, Germany). The inoculum was then diluted with either YPD broth (for TUN and FLC) or SD broth (for 5FC). Following this, 0.1 mL of the yeast suspension was added to each well of the 96-well microdilution plates, yielding a final inoculum concentration between 0.5 × 10³ and 2.5 × 10³ CFU/mL. The plates were subsequently incubated at 35°C. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration at which complete growth inhibition was observed. Three replicates were conducted for each drug concentration.

To generate mutants, we inoculated approximately 2.5 × 10³ cells/mL of the H99 strain into 1.5 mL of YPD broth containing a low concentration of 0.06 μg/mL of TUN. We performed three biological replicates. After 72 h of incubation with shaking, the cultures were washed and diluted with distilled water. We then spread approximately 300 cells onto YPD plates and incubated them at 30°C for 72 h. From each culture, we randomly selected 120 colonies and tested them for resistance to TUN using a spot assay.

To generate mutants, we suspended cells in distilled water and adjusted the concentration to 1 × 10⁷ cells/mL. We then spread 100 μL of this suspension onto YPD plates supplemented with a high concentration of 0.25 μg/mL TUN. The plates were incubated at 30°C for 5 days, allowing for the selection of resistant mutants. We randomly selected 30 mutants for further analysis and characterization. These 30 mutants were maintained in a −80°C freezer.

To investigate the genome instability of the aneuploid strain with disomies of Chromosomes 4 and 6, we performed the following steps: First, the strain with Chrs (4,6) x2 was thawed from the-80°C freezer and streaked onto YPD-agar plates, which were then incubated at 30°C for 72 h. From the resulting colonies, we randomly selected one small colony and suspended it in distilled water. We then diluted the cells in distilled water and spread approximately 200 cells onto a YPD plate, which was incubated at 30°C for 72 h. After incubation, we observed small (S), medium (M), and large (L) colonies. We randomly selected four colonies from each size category for further analysis.

DNA extraction, library construction, and sequencing were performed using previously described methods (Yang et al., 2021d). The data was then visualized using Ymap (Abbey et al., 2014). The raw fastq files were uploaded to Ymap (version 1.0)¹ and plotted as a function of chromosome position using the C. neoformans H99 reference genome (NCBI RefSeq assembly no. GCF_000149245.1).

Comparative analysis of strains: To investigate the transcriptome profiles of the strains, we performed RNA-seq as described previously with minor modifications (Ke et al., 2024). We first streaked the strains onto YPD plates from the −80°C freezer and incubated them at 30°C for 72 h. We then selected colonies with similar sizes, suspended them in a solution with an optical density (OD) of 0.1, and grew them in a shaker at 30°C until they reached an OD of 1.0. The cells were then collected by centrifugation and frozen in liquid nitrogen.

Treatment of H99 with TUN: We grew H99 in YPD broth in a shaker at 30°C from an OD of 0.1 to 1.0, and then divided the culture into two batches. One batch was treated with TUN at a final concentration of 0.25 μg/mL, while the other batch was treated with an equal volume of DMSO. Three hours later, the cells were collected by centrifugation and frozen in liquid nitrogen.

Total RNA extraction, purification, library construction, and sequencing were performed as described previously (Ke et al., 2024). We obtained three biological replicates for each strain and used DESeq2 (Love et al., 2014) to analyze the differential gene expression profiles. We considered genes with a False Discovery Rate (FDR)-adjusted p value <0.05 and expression fold changes of more than 1.5 or less than −1.5 as differentially expressed.

Significance analysis of differences between growth curves was performed using Tukey HSD (Honestly Significant Difference) test.

The sensitivity of the C. neoformans lab strain H99 to TUN was evaluated. In YPD broth, the minimum inhibitory concentration (MIC) of TUN was found to be 0.125 μg/mL. Additionally, TUN at a concentration of 0.06 μg/mL significantly inhibited the growth of H99 (p < 0.001, Tukey test), while TUN at a concentration of 0.03 μg/mL did not significantly inhibit growth (p > 0.05, Tukey test) (Figure 1A).

On YPD agar plates, H99 was able to grow in the presence of TUN at a concentration of 0.06 μg/mL, although the resulting colonies were smaller than those on the control plate. TUN at concentrations of 0.125 μg/mL and 0.25 μg/mL completely inhibited the growth of H99 (Figure 1B).

Based on these results, TUN at a concentration of 0.06 μg/mL in YPD broth was found to be sub-inhibitory to H99, while TUN at a concentration of 0.125 μg/mL on YPD agar (and in YPD broth) was inhibitory to H99.

We explored whether sub-MIC concentrations of TUN could select for adaptive mutants in the H99 strain. We cultured the strain in YPD broth supplemented with 0.06 μg/mL TUN, starting with a cell density of approximately 2.5 × 10³ cells/mL. After 72 h, we randomly selected 120 colonies from each culture and tested them for TUN susceptibility, with three biological replicates. Among these colonies, we identified five mutants that outperformed the parent strain (Figure 2A). The broth microdilution assay revealed that the parent strain H99 exhibited a MIC of 0.125 μg/mL for TUN, whereas the five mutants displayed MICs ranging from 0.5 to 1 μg/mL. Consequently, the five mutants demonstrated greater resistance to TUN compared to the parent strain.

Sequencing analysis of five selected mutants revealed diverse aneuploid karyotypes: Low-#1 had disomy of Chr12 (Chr12x2), Low-#2 had disomies of Chr9 and Chr12 (Chrs (9,12) x2), Low-#5 had disomies of Chr6 and Chr12 (Chrs (6,12) x2), and Low-#3 and Low-#4 had segmental disomy of the left arm of Chr4 (SegChr4x2) (Figure 2B). Thus, exposure to sub-inhibitory levels of TUN led to the selection of aneuploid TUN-resistant mutants with diverse karyotypes.

Variation analysis revealed few genetic mutations in the mutants (Supplementary Table S1). Low-#1 had a missense mutation in CNAG_06222, encoding the large subunit ribosomal protein L32e, while Low-#3 had a missense mutation in CNAG_00807, whose function is unknown.

We further examined whether the mutants displayed cross-resistance to antifungal drugs. Spot assays demonstrated that mutants with SegChr4x2 were more resistant to FLC than H99, while mutants with Chr12x2 and Chrs (9,12) x2 were more resistant to 5FC than H99 (Figure 2C). The broth microdilution assay indicated that the parent strain H99 had a MIC of 32 μg/mL for FLC, while the two mutants containing SegChr4x2 exhibited MICs greater than 128 μg/mL. For 5FC, H99 displayed an MIC of 0.5 μg/mL, whereas the mutants with Chr12x2 and Chrs (9,12) x2 had MICs of 4 μg/mL.

Thus, depending on the aneuploid chromosome, certain mutants exhibited cross-resistance to antifungals.

We plated approximately 1 million H99 cells on YPD plates containing 0.125 μg/mL and 0.25 μg/mL TUN, respectively. After 5 days, we observed lawn growth on the plate with 0.125 μg/mL TUN, while 92 colonies grew on the plate with 0.25 μg/mL TUN. We randomly selected 30 mutants from the latter plate for further analysis. Whole-genome sequencing revealed that 8 mutants had a normal euploid karyotype, while 22 mutants displayed diverse aneuploid karyotypes, consisting of 18 distinct patterns. Notably, 17 out of 22 aneuploids exhibited disomy or trisomy of Chromosomes 4 and 6, either alone or in combination with other chromosomes. Five aneuploids had either Chr4x2 or Chr6x2, but not both, along with disomy of 2–6 other chromosomes (Figure 3). This suggests that amplifications of Chr4 and Chr6, alone or in combination with other chromosomes, were the primary genomic changes among mutants selected under high TUN concentration.

We identified several genetic mutations in the mutants (Supplementary Table S1). For example, mutants high-#8 and high-#12 had the same frameshift mutation in gene CNAG_06130, while mutants high-#15, high-#17, and high-#26 had missense mutations in CNAG_05936, CNAG_01727, and CNAG_05396, respectively. Mutant high-#20 had a missense mutation in CNAG_04310 and a stop-gained mutation in CNAG_06382, while mutant high-#29 had missense mutations in CNAG_03862 and CNAG_03059. However, the functions of proteins encoded by CNAG_06130, CNAG_04310, CNAG_03862, and CNAG_03059 remain unknown.

We found that some mutants that grew in the presence of high concentrations of TUN also showed resistance to other antifungal drugs, including FLC and 5FC. To further investigate this, we tested the antifungal resistance of all these mutants using disk diffusion assays. Based on their resistance profiles, we categorized the mutants into four distinct groups (Figure 4).

One group of mutants was resistant to 5FC only and consisted of 8 mutants, which had MICs for 5FC ranging from 1 to 8 μg/mL. Interestingly, three of these mutants had a normal euploid karyotype, while the remaining five had amplifications of at least three chromosomes.

Another group of mutants was resistant to FLC only and consisted of three mutants, each having an MIC of 64 μg/mL for FLC. Each of these mutants had distinct chromosomal changes, including disomy of Chromosomes 4 and 6, disomy of Chromosome 4 and trisomy of Chromosome 6, and disomy of Chromosomes 4, 6, and 12.

A third group of mutants showed cross-resistance to both 5FC and FLC, and consisted of only one mutant. The MICs for FLC and 5FC were 128 μg/mL and 8 μg/mL, respectively. This mutant had a complex karyotype, with disomy of Chromosomes 1, 4, 6, and 12.

The final group of mutants did not show obvious resistance to either 5FC or FLC, and consisted of 18 mutants. However, some of these mutants were hypersensitive to FLC, while others were hypersensitive to 5FC.

A mutant with a chromosomal abnormality, Chrs (4,6) x2, was analyzed for its physical characteristics and genetic stability. To do this, around 400 cells of the mutant were grown on a special agar plate (Figure 5A). After two days, differences in the size of the resulting colonies were observed (Figure 5A). The area of each colony was then measured (Figure 5B). Colonies that were smaller than 0.03 cm² were classified as small, those between 0.035 cm² and 0.04 cm² as medium, and those larger than 0.045 cm² as large (Figure 5A,B). Four colonies from each size group were randomly selected for further genetic analysis. The results showed that all four small colonies still had the Chrs (4,6) x2 abnormality, while the four medium colonies had a different abnormality, Chr4x2, and the four large colonies had a normal chromosome count (Figure 5C). This suggests that the Chrs (4,6) x2 mutant is unstable and may tend to lose the extra copy of chromosome 6 or all extra chromosomes.

Randomly selected S, M, and L colonies, one from each type, were compared to the H99 strain for resistance to TUN. The spot assay indicated that both S and M colonies exhibited resistance, while the L colony did not (Supplementary Figure S1). Thus, the presence of an additional copy of Chr4 alone was sufficient to confer resistance to TUN, and the loss of all extra copies of aneuploid chromosomes resulted in a loss of TUN resistance.

Our study reveals that disomy of Chrs (4,6), either alone or in combination with disomy of other chromosomes, was the primary genomic alteration among the mutants. Additionally, Chrs (4,6) x2 conferred resistance not only to TUN but also to FLC. To elucidate why Chrs (4,6) x2 led to cross-resistance to both drugs, we compared the transcriptome of one Chrs (4,6) x2 mutant to that of the wild type (Figure 6).

Transcription of 392 genes was detected on Chr4, with 312 genes showing ratios higher than 1.3. Similarly, transcription of 533 genes was detected on Chr6, with 442 genes having ratios higher than 1.3. Consequently, 79.6 and 82.9% of transcribed genes on Chr4 and Chr6, respectively, were up-regulated at least 1.3 times in the Chrs (4,6) x2 strain compared to H99. In contrast, on euploid chromosomes, only 7.5–12.1% of genes were up-regulated to this extent.

Overall, 910 genes were up-regulated across the genome, with 267 and 388 genes on Chr4 and Chr6, respectively. Conversely, 990 genes were down-regulated, with only 8 and 4 on Chr4 and Chr6, respectively. Thus, 72.0% (655 out of 910) of the up-regulated and 1.2% (12 out of 990) of the down-regulated genes were located on aneuploid chromosomes. Consequently, disomy of Chr4 and Chr6 generally led to increased expression of genes on the aneuploid chromosomes and altered expression of genes on euploid chromosomes.

Gene ontology (GO) analysis revealed that 21 genes involved in response to TUN (GOID: 1904576) were significantly enriched among the up-regulated genes, with 14 of them located on Chr4 or Chr6 (Supplementary Table S2). Notably, among the genes encoding efflux pumps, AFR1/CNAG_00730, MDR1/CNAG_00796, and PDR16/CNAG_04984 were up-regulated, with AFR1 and MDR1 on Chr1 and PDR16 on Chr4. Among the ERG genes, only ERG19/CNAG_05125 and ERG20/CNAG_02084 were up-regulated, with ERG19 on Chr4 and ERG20 on Chr6. Conversely, ERG4/CNAG_02830 was down-regulated (Supplementary Table S2). However, other ERG genes, such as ERG11/CNAG_00040, ERG3/CNAG_00519, or ERG6/CNAG_03819, showed no differential expression in Chrs (4,6) x2 compared to H99. Thus, we propose that Chrs (4,6) x2 confers resistance to TUN and FLC by simultaneously up-regulating multiple genes on both euploid and aneuploid chromosomes.

We investigated whether TUN also induced antifungal resistance in clinical isolates. Approximately 1 million cells of the clinical isolate GLS#4458 were spread on YPD-agar plates supplemented with TUN. After 5 days, the plate with 0.125 μg/mL TUN showed lawn growth, while the plate with 0.25 μg/mL TUN displayed a few 100 visible colonies (Figure 7A). Sixteen mutants (#1–#16) were randomly selected. Spot assays revealed that all of them were able to grow in the presence of 0.25 μg/mL TUN, whereas growth of the wild-type GLS#4458 was completely inhibited (Figure 7B), indicating that all the mutants developed resistance to TUN. Disk assays were conducted using disks containing 200 μg FLC, and three mutants, #1, #9, and #12, exhibited smaller inhibition zones compared to the parent strain (Figure 7C), suggesting that these three mutants acquired resistance to FLC.