The strains used in this study are listed in Supplementary Table 1. C. parapsilosis clinical isolate #12108 was used as the wild-type strain. The stock culture was preserved in 25% of glycerol and maintained at −80°C. Cells were routinely grown in the yeast extract–peptone–dextrose (YPD) media (1% [w/v] yeast extract, 2% [w/v] peptone, and 2% [w/v] D-glucose) at 37°C in a shaking incubator at 150–200 rpm. For solid medium, 2% [w/v] agar was added. SD agar plates (0.67% [wt/vol] yeast nitrogen base without amino acids, 2% [wt/vol] D-glucose, and 2% [wt/vol] agar) were used for testing tolerance to 5FC. Drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. The concentrations of echinocandins, 5FC, and FLC were 10, 0.5, and 40 mg/ml, respectively.

Cells were suspended in distilled water. Cell density was determined by using a hemocytometer and was adjusted to 1 × 10⁷ cells/ml. A total of 100 μl of cell suspension were spread on YPD plates supplemented with 100, 200, and 400 ng/ml of CSP. On day 5, only ~368 colonies (adaptors) appeared on the plate with 400 ng/ml of CSP, while on other plates, we saw lawn growth. A total of 30 adaptors (TJ60–TJ89) were randomly chosen from the plate with 400 ng/ml of CSP. Each adaptor was streaked from the drug plate onto the YPD plate. The plates were incubated at 37°C for 3 days. Four to six colonies with similar sizes were selected and frozen in 1 ml of 25% glycerol at −80°C.

Cells were suspended in distilled water and adjusted to 1 × 10⁷ cells/ml. A total of 3 μl of 10-fold serial dilutions were spotted on YPD or SD plates with or without drugs (control) at 37°C and photographed after 3 days.

Cells were suspended in YPD broth. Cell densities were adjusted to 2.5 × 10³ cells/ml in YPD broth with or without test drugs in a 96-well plate. The plate was incubated at 37°C. OD₅₉₅ was monitored in a Tecan plate reader (Infinite F200 PRO, Tecan, Switzerland) at 15 min time intervals for 48 h. Data are represented as the mean ± SD of three biological replicates.

As described previously (Yang et al., 2021d), Chr1x3 and Chr5x3 adaptors were streaked from−80°C freezer to YPD agar and incubated at 37°C for 72 h. One small colony was randomly chosen and suspended in distilled water. Cells were diluted with distilled water and ~200 cells were spread on a YPD plate and incubated at 37°C for 72 h. One small (S) colony and one large (L) colony were randomly chosen for further studies.

The CLSI M44-A2 guidelines (CLSI, 2012) for the antifungal disk diffusion susceptibility testing were followed with slight modifications. Strains were grown on agar plates, and cell density was adjusted to 1 × 10⁶ cells/ml as described earlier. A total of 100 μl of cell suspension was plated on plates. One paper disk (GE Healthcare, USA) was placed in the center of each plate. The plates were then incubated for 72 h and photographed.

The test strains were grown on YPD plates at 37°C at a density of ~200 colonies per plate. Colonies were collected by centrifugation in a microfuge at 3,000 rpm for 1 min. Genomic DNA was extracted manually using the phenol–chloroform method (Selmecki et al., 2015). The genomic DNA library was prepared by BGI (Wuhan, China) according to their standard preparation protocol. Approximately 1 μg of genomic DNA was randomly fragmented with a Covaris LE220. Fragments (300–400 bp) were selected, end-repaired, and 3' adenylated using the Agencourt AMPure XP-Medium kit, then ligated to adaptors. The ligation products were amplified by PCR. After purification, the PCR products were heat denatured, then circularized with a splint oligo sequence. The single-strand circular DNA (ssCirDNA) was formatted as the final library, qualified by QC, and then sequenced by BGISEQ-500. ssCir DNA molecules formed a DNA nanoball (DNB) containing more than 300 copies through rolling-cycle replication. The DNBs were loaded into a patterned nano array by using high-density DNA nanochip technology and were sequenced on the BGISEQ-500 platform using BGISEQ-500 high-throughput sequencing kit (PE100). Finally, pair-end 100 bp reads were obtained by combinational probe-anchor synthesis (cPAS). Raw FASTQ files were uploaded to YMAP (version 1.0) (http://lovelace.cs.umn.edu/Ymap/) (Abbey et al., 2014). Read depth was plotted as a function of chromosome position using the CDC317 reference genome of C. parapsilosis (http://www.candidagenome.org/download/sequence/C_parapsilosis_CDC317/current/). Contig005504, contig005569, contig005806, contig005807, contig005809, contig006110, contig006139, and contig006372 were renamed to chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, and chromosome 8, respectively. Chromosome end bias and GC content bias were corrected by YMAP (Abbey et al., 2014).

RNA-seq was performed as described previously (Yang et al., 2021b). Strains were streaked onto YPD plates from the −80°C freezer. After 72 h of incubation at 37°C, several colonies of similar sizes were chosen. Colonies were suspended in distilled water and adjusted to 1 × 10⁴ cells/ml. A total of 100 μl of cell suspension were spread on YPD plates. The plates were incubated at 37°C for 72 h. Cells were collected by centrifugation, washed, and flash-frozen in liquid nitrogen. The total RNA was extracted for nine independent samples, corresponding to three conditions and three biological replicates. Total RNA extraction and purification, library construction, and sequencing were performed as described in Yang et al. (2013). Raw sequence files (.fastq files) underwent quality control analysis using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were mapped to the C. parapsilosis CDC317 reference genome (http://www.candidagenome.org/download/sequence/C_parapsilosis_CDC317/current/). Differential gene expression profiling was carried out using DESeq2 (Love et al., 2014) with standard parameters. Genes with false discovery rate (FDR)-adjusted P-value (<0.05) and expression fold changes of more than 1.3 or <-1.3 were considered differentially expressed.

Cells were grown under the same experimental conditions as in RNA-seq. RT-qPCR was performed in 96-well plates (Bio-Rad) on the CFX Touch 96-well Real-Time Systems (Bio-Rad). Primer sequences are listed in Supplementary Table 2. The reaction mix was performed using 5 μl of iTaq Universal SYBR Green Supermix (Bio-Rad), 2 μl of 2 μM primer mix, 2 μl of a diluted 1:10 cDNA, and water to make up the final volume to 10 μl. Cycling conditions were 95 °C for 3 min, 40 cycles of 95 °C for 5 s, and 60 °C for 30 min. Melt curve analysis conditions were 5 s at 95°C and then 5 s each at 0.5°C increments between 60°C and 95°C. ACT1 (CPAR2_201570) was the internal control. Fold change was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). All RT-PCR experiments were performed using three biological and three technical replicates.

The significance of differences between growth curves was performed using Tukey's honest significant difference (HSD) test.

In order to obtain CSP adaptors, ~1 million cells of C. parapsilosis clinical isolate #12108 were spread on the YPD plate supplemented with 400 ng/ml of CSP. After 5 days of incubation at 37°C, ~368 colonies (adaptors) were obviously visible on the plate (Figure 1A). We randomly picked up 30 adaptors (TJ60–TJ89, Supplementary Figure 1) with different colony sizes on the drug plate. Whole genome sequencing indicated that all the adaptors were aneuploid: 29 had trisomy of chromosome 5 (Chr5x3), and 1 (TJ74) had trisomy of chromosome 1 (Chr1x3) (Figure 1B). Of note, there are three FKS genes in C. parapsilosis genome: GSC1/CPAR2_106400, GSL1/CPAR2_109680, and GSL2/CPAR2_804030. Sequences of these three FKS genes were visualized in Integrative Genomics Viewer (IGV), and we did not detect any mutation in the 30 adaptors. GSC1 and GSL1 are on Chr2, and GSL2 is on Chr4, but none of the adaptors had aneuploidy of Chr2 or Chr4. Therefore, Chr5x3 was the major mechanism of adaptation to CSP. Mutations of FKS genes and aneuploidy of chromosomes on which the FKS genes reside were not detected.

It is known that in Saccharomyces cerevisiae and C. albicans, aneuploids usually have fitness loss in rich medium in the absence of stress (Pavelka et al., 2010; Yang et al., 2021d). In C. albicans, gain-of-function mutations of genes associated with resistance to azoles also have fitness costs in vitro and in vivo (Sasse et al., 2012; Hill et al., 2015; Popp et al., 2017). We asked if the aneuploid CSP adaptors also had fitness loss. Growth curves of the adaptors and the parent were measured in YPD broth (Figure 2A). The Chr5x3 adaptor TJ60 and the Chr1x3 adaptor TJ74 were significantly less fit than the parent (p < 0.0001 and p < 0.05, respectively. Tukey's HSD test).

We investigated whether the adaptors gained resistance or tolerance to CSP. DDAs with disks containing CSP were performed. In DDAs, the radius of the zone of inhibition (RAD) is reverse proportional to MIC (Milici et al., 2007). Here, we found that none of the adaptors had an obvious change in RAD as compared to the parent (Figure 2B). diskImageR analysis of DDA pictures indicated the parent, the Chr1x3 adaptor, and the Chr5x1 adaptor had the same RAD value of 9.5 ± 0.7. When tested with another echinocandin drug micafungin (MCF), the parent and the adaptors had the same RAD value of 12.0 ± 0.0. Therefore, the CSP adaptors did not develop resistance to CSP or MCF (Figure 2B). This is consistent with the finding that none of the adaptors had mutations of FKS genes, since FKS mutations in clinical isolates of Candida spp. usually cause increased resistance to CSP (Balashov et al., 2006; Garcia-Effron et al., 2010; Imtiaz et al., 2012; Beyda et al., 2014; Marti-Carrizosa et al., 2015; Naicker et al., 2016; Szymankiewicz et al., 2021).

To confirm the development of CSP resistance and FKS mutations were rare events, we repeated this experiment by testing more adaptors. Approximately 1 million cells of #12108 were spread on YPD plates supplemented with 400 ng/ml of CSP. Randomly 192 were tested with DDAs and none of them had reduced RAD (data not shown).

In C. albicans, strains tolerant to azole drugs fluconazole (FLC) (Rosenberg et al., 2018) or ketoconazole (Xu et al., 2021) have increased FoG₂₀ when tested by DDA with disks containing azoles, as indicated by colonies growing inside of the zone of inhibition. However, here we found none of the CSP adaptors had colonies growing inside of the zone of inhibition (Figure 2B). Therefore, the CSP adaptors do not have reduced RAD₂₀ or increased FoG₂₀.

Previously we found spot assay was a sensitive method to detect improved ability to grow in the presence of CSP in C. albicans (Yang et al., 2019). Here, we investigated whether spot assay could detect CSP tolerance in the adaptors. Spot assay indicated that the Chr5x3 adaptor (TJ60) grew better than the parent in the presence of CSP and MCF. Furthermore, spot assay indicated that all 29 Chr5x3 adaptors grew better than the parent in the presence of CSP and MCF (Supplementary Figure 1). However, the Chr1x3 adaptor (TJ74) did not grow better than the parent (Figure 2C). Thus, all CSP adaptors were aneuploid and had fitness cost in rich medium in the absence of stress; however, Chr5x3 enabled better fitness in the presence of CSP and MCF.

Previously we found aneuploidy caused cross-tolerance to unrelated stresses in C. albicans (Yang et al., 2013, 2019, 2021b), C. parapsilosis (Yang et al., 2021c), and Cryptococcus neoformans (Yang et al., 2021a). Here, we investigated whether the aneuploid CSP adaptors caused cross-tolerance to other antifungal drugs. One Chr5x3 adaptor (TJ60) and one Chr1x3 adaptor (TJ74) were compared to the parent. DDAs with disks containing FLC or 5FC were performed.

When tested with FLC, the parent, Chr5x3 adaptor and Chr1x3 adaptor had a RAD₂₀ of 17.0 ± 0.0, 23.5 ± 0.7, and 18.5 ± 0.7, respectively (Figure 3A). Therefore, Chr5x3 caused hypersensitivity to FLC, and Chr1x3 caused slightly increased sensitivity to FLC. When tested with 5FC, the parent, Chr5x3 adaptor, and Chr1x3 adaptor did not show an obvious change in RAD. However, inside the zone of inhibition, the Chr5x3 adaptor had lawn growth, while the Chr1x3 adaptor and the parent exhibited clear zones (Figure 3A). We investigated to what extent Chr5x3 can tolerate 5FC. Spot assay indicated that the growth of both parent and Chr1x3 was completely inhibited by 0.25 μg/ml of 5FC on the SD plate, but Chr5x3 could tolerate up to 128 μg/ml of 5FC (Figure 3B). Therefore, Chr5x3 caused hypersensitivity to FLC and tolerance to 5FC.

We asked if aneuploids in C. parapsilosis were unstable. Approximately 200 cells of the Chr5x3 adaptor (TJ60) and Chr1x3 adaptor (TJ74) were spread on YPD plates. After incubation at 37°C for 48h, both adaptors exhibited colony instability: most colonies were small (indicated by cyan arrows in Figure 4A) and a few colonies were large (indicated by magenta arrows in Figure 4A). Whole-genome sequencing indicated that the large colonies were euploids (Figure 4B). Spot assay indicated that only the small colony of the Chr5x3 adaptor was tolerant to CSP, MCF, and 5FC. The large colony was not tolerant (Figure 4C). We investigated whether all 30 adaptors (TJ60–TJ89) were unstable. We found that all of them yielded small and large colonies on YPD plates (data not shown). Spot assay indicated that none of the large colonies was tolerant to CSP (Supplementary Figure 2). Therefore, in C. parapsilosis, aneuploids (Chr5x3 and Chr1x3) are unstable. Increased copy number of Chr5 causes tolerance to echinocandins and cross-tolerance to 5FC. Reversion of Chr5x3 to Chr5x2 is accompanied by the loss of tolerance and cross-tolerance to antifungal drugs.

Since the CSP adaptors do not have FKS mutations, and FKS genes are not on the aneuploid chromosome, we investigated the mechanism of drug tolerance by performing RNA-seq and we compared the transcriptomes of CSP adaptors to the parent. Among the 1412 ORFs on Chr5, 744 were differentially expressed genes (q < 0.05) in the Chr5x3 adaptor as compared to the parent. Only 14 of the 744 ORFs were downregulated. All the remaining ORFs (98.1%) were upregulated. Among the 375 ORFs on Chr1, 253 were differentially expressed genes (q < 0.05) in the Chr1x3 adaptors as compared to the parent. All of them (100%) were upregulated. Therefore, both Chr1x3 and Chr5x1 caused proportional elevated transcription of the genes on the aneuploid chromosomes (Figure 5A).

Among genes upregulated in the Chr5x3 adaptor, processes associated with peroxisome, DNA repair, cell cycle, and catabolism were significantly enriched. In contrast, among genes upregulated in the Chr1x3 adaptor, processes associated with biosynthesis including organic substances, cellular nitrogen compounds, and macromolecules were significantly enriched (Supplementary Table 3). Several processes were commonly downregulated in Chr5x3 and Chr1x3 adaptors, including protein folding (GOID: 0006457), mitochondrion organization (GODI: 0007005), and protein import into mitochondrial intermembrane space (GOID: 0045041). Some GO terms were significantly enriched in Chr5x3 downregulated genes and enriched in Chr1x3 upregulated genes, including ribosome biogenesis (GOID: 0042254), ribosome localization (GOID: 0033750), ribosomal subunit export from the nucleus (GOID: 0000054), peptide biosynthetic process (GOID:0043043), and translation (GOID: 0006412) (Figure 5B).

We investigated whether genes associated with β-1,3-glucan synthesis and chitin synthesis and degradation were differentially regulated in the Chr5x3 adaptor TJ60. None of the FKS genes (GSC1, GSL1, and GSL2) was upregulated in the Chr5x3 adaptor. Among the genes encoding chitin synthase (CHS1/CPAR2_800050, CHS2/CPAR2_701490, CHS3/CPAR2_801800, CHS4/CPAR2_807030, CHS5/CPAR2_210990, CHS7/CPAR2_212710, and CHS8/CPAR2_502940), only one gene, CHS7 was significantly upregulated in the Chr5x3 adaptor. CHS7 is on Chr5. Among the genes encoding chitinases (CHT2/CPAR2_502140, CHT3/CPAR2_200660, and CHT4/CPAR2_211950), two genes, CHT3 and CHT4 are on Chr5 but their transcription was compensated to the disomic level (Supplementary Table 4). Therefore, we posit that Chr5x3 enables tolerance to CSP via increasing copy number and transcription of CHS7 and reducing the transcription of CHT3 and CHT4, thereby increasing chitin content in the cell wall.

We also investigated whether genes associated with 5FC tolerance were downregulated in the Chr5x3 adaptor. FUR1 was significantly downregulated in the Chr5x3 adaptor. FCA1 and FCY2 did not exhibit obvious change (Supplementary Table 4). FUR1 is on Chr7. We posit that Chr5x3 causes 5FC tolerance via indirectly downregulating the transcription of the FUR1 gene on the euploid chromosome.

Compared to parent, several ERG genes were downregulated in Chr5x3 adaptor, but not in Chr1x3 adaptor, including ERG3/CPAR2_105550, ERG5/CPAR2_703970, ERG8/CPAR2_400710, and ERG11/CPAR2_303740. ERG11 was upregulated in the Chr1x3 adaptor. ERG1/CPAR2_210480, ERG4/CPAR2_502980, and ERG13/CPAR2_701400 were upregulated in the Chr1x3 adaptor, but not in the Chr5x3 adaptor. ERG12/CPAR2_803530 was downregulated in both Chr1x3 and Chr5x3 adaptors. UPC2/CPAR2_207280 encodes a transcription factor that positively regulates ergosterol biosynthetic genes. In Chr5x3 but not in the Chr1x3 adaptor, UPC2 was upregulated. In addition, CDR1/CPAR2_405290, which encodes the drug efflux pump, was downregulated in the Chr5x3 adaptor, but not in the Chr1x3 adaptor, as compared to the parent (Supplementary Table 4). Taken together, we posit that Chr5x3 causes hypersensitivity to FLC via decreasing the expression of ERG genes and CDR1.

We visualized the genome sequencing data of the Chr5x3 adaptor and the parent in IGV, and we did not see mutations of genes ERG3, ERG11, or CDR1

The expression profile of genes in RNA-seq results was validated by reverse transcriptase PCR (RT-PCR). Six genes were tested, including CHS7, CHT3, CHT4, FUR1, ERG11, and CDR1 (Supplementary Figure 4).

We investigated how a Chr1x3 strain would adapt to CSP. Approximately one million cells of adaptor TJ74 were spread on the YPD plate supplemented with 400 ng/ml of CSP. Randomly 30 adaptors (TJ2267–TJ2296) were chosen. Spot assay indicated that 22 of the 30 adaptors grew better than TJ74 and #12108 in the presence of CSP (Supplementary Figure 3). We sequenced all 22 tolerant adaptors. Based on the karyotypes, the tolerant adaptors were categorized into four classes: Class 1 adaptors (n = 17) had Chr5x3 alone. Class 2 adaptor (n = 1) had Chr5x3, segmental trisomy of Chr3 (SegChr3x3, from 0.43 Mb till the right telomere), and segmental trisomy of Chr8 (SegChr8x3, from left telomere till 0.87 Mb). Class 3 adaptor (n = 1) had Chr5x3, segmental trisomy of Chr1 (from 0.40 Mb till the right telomere), and segmental trisomy of Chr2 (SegChr2x3, from left telomere to 0.25 Mb). Class 4 adaptors (n = 3) had Chr1x3, SegChr3x3, and SegChr8x3 (Figure 6A). Of note, none of the 22 tolerant adaptors had FKS mutations. Although GSC1 and GSL1 are on Chr2, in the Class 3 adaptor, which had SegChr2x3, the amplified region of Chr2 did not encompass GSC1 and GSL1. Therefore, the Chr1x3 strain adapted to CSP mainly via losing Chr1x3 and gaining Chr5x3 (19 out of 22) or maintaining Chr1x3 but gaining SegChr3x3 and SegChr8x3. Genetic mutation or elevated copy number of FKS genes did not happen.

We enquired whether Chr1x3-derived CSP adaptors were cross-tolerant to 5FC. DDAs on SD plates with paper disks containing 5FC indicated that all Class 1, Class 2, and Class 3 adaptors were tolerant to 5FC and Class 4 adaptors were not (Figure 6B). Therefore, adaptors with Chr5x3 were cross-tolerant to CSP and 5FC. Adaptors without Chr5x3 were only tolerant to CSP but not tolerant to 5FC.