The C. glabrata FLC-tolerant isolates CG4 and CG8, and the non-tolerant reference strain BG2 served as the progenitors for this study. The profile of FLC tolerance in CG4 has been detailed in our previous report (Zheng et al., 2024b). Stock cultures were preserved in 25% glycerol and stored at −80°C. Cells were routinely cultured in Yeast Extract-Peptone-Dextrose (YPD) medium, which contains 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose, at 30°C using a shaking incubator set to 150–200 rpm. For YPG medium, the composition included 1% (w/v) yeast extract, 0.2% (w/v) peptone, and 3% (w/v) glycerol, with 2% (w/v) agar added for solid media. Drug solutions were prepared in dimethyl sulfoxide (DMSO) and stored at −20°C.

Disk diffusion assays were performed according to the protocols outlined in our previous studies (Guo et al., 2024; Zheng et al., 2024b; Zheng et al., 2024c), following the CLSI M44-A2 guidelines for antifungal disk diffusion susceptibility testing (CLSI, 2009), with minor modifications. Briefly, strains were streaked from glycerol stocks onto YPD agar plates and incubated at 30°C for 48 h. Colonies were then suspended in distilled water and adjusted to a concentration of 1 × 10⁶ cells/mL. A volume of 100 μL of this cell suspension was evenly spread across YPD plates. An empty paper disk (6 mm diameter and 0.7 mm thickness) was saturated with 5 μL of 40 mg/mL FLC and placed at the center of each plate. The plates were subsequently incubated at 30°C and photographed after 48 h. The analysis of the disk diffusion assay was conducted using the diskImageR pipeline (Gerstein et al., 2016), measuring parameters such as the fraction of growth within the zone of inhibition (FoG) and the radius of inhibition (RAD).

For the isolation process, cells were suspended in distilled water and carefully adjusted to a concentration of 1 × 10⁶ cells/mL. Subsequently, 100 μL of this cell suspension was evenly spread onto a YPD plate supplemented with 1 μg/mL TUN. An empty paper disk saturated with 5 μL of 40 mg/mL FLC was placed at the center of the plate.

Following an incubation period of 48 h at 30°C, four colonies were randomly selected from within the ZOI for further examination. These chosen colonies were streaked onto fresh YPD plates and underwent an additional 48-h incubation. From each replicate, a single colony was then meticulously chosen to progress to the subsequent stage of meticulous analysis and exploration.

The cells were suspended in distilled water and adjusted to a concentration of 1 × 10⁷ cells/mL. Subsequently, 100 μL of this cell suspension was evenly spread on YPD plates supplemented with TUN. The plates were then incubated at 30°C for a duration of 5 days, after which adaptors were randomly chosen from the drug-treated plates.

Cells were suspended in distilled water and adjusted to a concentration of 1 × 10⁷ cells/mL. A volume of 3 μL of the cell suspension was spotted onto YPD or YPG plates. For testing susceptibility to TUN, 3 μL of 10-fold serial dilutions were spotted on YPD plates containing 8 μg/mL TUN. The plates were incubated at 30°C and photographed after 48 h.

The technique for inducing petite formation with Ethidium bromide (EtBr) was adapted from Fox et al. (1991) with slight modifications. Thawed test strains were streaked onto YPD plates and incubated at 30°C for 48 h. A single colony was then inoculated into YPD broth with 25 μg/mL EtBr, followed by transfer to a second culture with the same medium. Saturated cultures were streaked onto YPD plates to isolate colonies, which were subsequently streaked onto YPD and YPG plates to confirm respiratory deficiency.

To compare between progenitor and petite strains, they were cultured in YPD broth until reaching the logarithmic phase (OD₆₀₀ = 1.0). To assess the effect of TUN on gene expression, the logarithmic phase cultures were split into two groups. One group received 8 μg/mL TUN supplementation, while the other was supplemented with an equivalent amount of vehicle. After a 3-h incubation period, the cells were harvested by centrifugation.

Total RNA was extracted using YeaStar RNA kit (Zymo Research) following the manufacturer’s guidelines. The RNA concentration and purity were evaluated with a spectrophotometer (NanoDrop 2000C; ThermoFisher Scientific) through absorbance measurements at 230 nm (OD₂₃₀), 260 nm (OD₂₆₀), and 280 nm (OD₂₈₀). Additionally, RNA integrity was confirmed by electrophoresis on 1% denaturing and non-denaturing agarose gels in selected samples.

The RNA samples were treated with DNase I (ThermoFisher Scientific) at 37°C for 30 min following the manufacturer’s protocol. Approximately 1 μg of total RNA underwent reverse transcription (RT) using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

The expression of candidate genes was quantified by real-time RT-PCR using the CFX96 Touch Real-Time PCR system (Bio-Rad). The housekeeping ACT1 was used as internal control. The relative quantification of gene expression was performed by the 2^−ΔΔCT method (Schmittgen and Livak, 2008). Each reaction was performed in triplicate, and mean values of relative expression were determined for each gene. Primers are listed in Supplementary Table S1.

The experiment was performed according to the Clinical and Laboratory Standards Institute (CLSI) recommendations (CLSI, 2017) with slight modifications. Briefly, yeast cells were harvested during the logarithmic growth phase, washed twice with sterile distilled water, and resuspended in distilled water. The cell density was adjusted to a final concentration of 2.5 × 10³ cells/mL in YPD broth supplemented with fluconazole (FLC) at concentrations ranging from 0.125 to 128 μg/mL. The cell suspensions were then aliquoted into 96-well microtiter plates, with each well containing 200 μL of the suspension. The plates were incubated at 30°C for 24 h under static conditions. After incubation, the optical density at 600 nm (OD600) was measured using a microplate reader to quantify cell growth. Each condition was tested in triplicate to ensure reproducibility, and control wells containing YPD broth without FLC were included to account for background growth.

Multilocus sequence typing (MLST) analysis was conducted as previously described by Dodgson et al. (2003). Six loci (FKS, LEU2, NMT1, TRP1, UGP1, and URA3) were amplified using the primers specified in Dodgson et al. (2003). PCR reactions were carried out in 20-μL volumes containing 5 ng of genomic DNA, 10 μL of 2 × Phusion Green Hot Start II High-Fidelity PCR Master Mix (Fisher Scientific), and 0.1 μM of each primer. The amplified products were sequenced bidirectionally (forward and reverse) using the same primers as those employed for the PCR amplification.

All disk diffusion assays represent the average of three technical replicates, with error bars indicating the standard deviation. Statistical analyses were conducted using a two-tailed Student’s t-test in Microsoft Excel. A p-value of less than 0.05 was considered statistically significant. ** indicates p < 0.01, and *** indicates p < 0.001.

In our study, we evaluated the effects of TUN on the FLC tolerance of three clinical isolates of C. glabrata. Each isolate exhibited a notable tolerance to FLC, as evidenced by the presence of significant lawn growth within ZOI. In a pilot experiment, we tested the effect of various concentrations of TUN on FLC tolerance in CG4. We found that 0.5 μg/mL of TUN did not abolish FLC tolerance, whereas 1 and 2 μg/mL concentrations effectively eliminated FLC tolerance. At 4 μg/mL, the growth of most cells on the plate was inhibited (Supplementary Figure S1). Based on these results, we selected 1 μg/mL of TUN to assess its impact on FLC tolerance in CG4, as well as in two other C. glabrata isolates, CG8 and CG10.

Upon the supplementation of 1 μg/mL TUN, a marked change in the response was observed in all the 3 isolates: the ZOI became clear, indicating a loss of FLC tolerance (Figure 1, top panel).

Quantitative assessments revealed that a concentration of TUN at 1 μg/mL led to a significant decrease of FoG₂₀ values across all three tested isolates, with statistical significance confirmed (p < 0.001, two-tailed Student’s t-test). Interestingly, while TUN effectively diminished FLC tolerance, it did not appear to affect the RAD₂₀ (Figure 1, bottom panel), indicating that TUN’s mechanism of action primarily targets pathways associated with tolerance rather than directly impacting resistance mechanisms.

While TUN effectively abolished FLC tolerance in our experiments, we noted an intriguing phenomenon during the testing of the CG4 isolate; a few exceptionally large colonies were observed within ZOI, as indicated by red arrows in Figure 2A. To further investigate this anomaly, we randomly selected four of these colonies, designated as #1 through #4, for analysis.

Interestingly, although the progenitor strain CG4 was capable of growing on YPG plate—where glycerol served as the carbon source—none of the four chosen colonies exhibited growth under the same conditions. This observation suggests that these colonies may be petites, which are characterized by respiratory deficiencies due to defects in mitochondrial function (Figure 2B).

To assess the impact of TUN on these four colonies, we conducted a spot assay, which revealed that each of the four colonies demonstrated improved growth compared to the parental CG4 strain when exposed to TUN (Figure 2C). This finding indicates a possible adaptive response or compensatory mechanism in the petites that enhances their proliferation in the presence of TUN.

Furthermore, results from a disk diffusion assay using disks containing FLC showed that all four colonies exhibited clear ZOI with reduced overall size (Figure 2D). Quantitative analysis of the disk diffusion assay images confirmed that all four colonies had significantly lower values for both FoG₂₀ and RAD₂₀, with statistical significance denoted (p < 0.001, two-tailed Student’s t-test). These results collectively indicate that the four colonies not only lost FLC tolerance but also gained FLC resistance (Figure 2E).

In the experiments described above, the progenitor strain CG4 was exposed to a combination of TUN and FLC. In this section, we investigate the impact of TUN alone on the CG4 isolate. To assess this, CG4 cells were spread on YPD plates containing varying concentrations of TUN. Notably, on the plate with 16 μg/mL TUN, several hundred colonies emerged, which we refer to as “adaptors.” In contrast, lower concentrations of TUN resulted in a uniform lawn growth across the plates (Figure 3A).

From the colonies that appeared at the highest concentration, we randomly selected 16 adaptors for further analysis. These adaptors were then compared to the progenitor strain in terms of their resistance to TUN. A spot assay demonstrated that all 16 adaptors were capable of growing in the presence of 8 μg/mL TUN, while the progenitor strain exhibited marked inhibition of growth under the same conditions (Figure 3B). Thus, all the 16 adaptors gained resistance to TUN.

Next, we evaluated the ability of these adaptors to utilize glycerol as a carbon source, which is indicative of respiratory competency. Of the 16 adaptors tested, four—specifically #9, #10, #12, and #16—were able to grow on YPG plates, suggesting that the majority of the adaptors (12 out of 16) exhibited respiratory deficiencies (Figure 3C).

Lastly, we assessed the susceptibility of the adaptors to FLC. The petite adaptors demonstrated significantly RAD₂₀ and FoG₂₀ compared to the progenitor strain, indicating that they had lost FLC tolerance and gained FLC resistance. In contrast, the non-petite adaptors did not show significant changes in RAD₂₀ or FoG₂₀ when compared to the progenitor (Figure 3D).

Susceptibility to FLC was further assessed by determining the minimum inhibitory concentration (MIC). The progenitor strain and non-petite TUN adaptors exhibited MICs of 16 μg/mL, whereas the petite TUN adaptors showed significantly higher MICs, ranging from 64 to 128 μg/mL.

Besides CG4, another isolate, CG8, was also tested. We found that TUN at a concentration of 8 μg/mL significantly inhibited the growth of CG8 (Supplementary Figure S2A). Twelve adaptors (#1-#12) were randomly selected, and three of them (#3, #8, and #9) failed to grow on YPG plates (Supplementary Figure S2B). Disk diffusion assays showed that the petite adaptors had significantly lower FoG₂₀ and smaller RAD₂₀ values compared to the wild-type (p < 0.001, two-tailed Student’s t-test), while the non-petite adaptors did not exhibit significant changes in FoG₂₀ or RAD₂₀ (p > 0.05, two-tailed Student’s t-test).

In addition to the two clinical isolates, the reference strain BG2 was also tested. We found that TUN at 8 μg/mL significantly inhibited BG2 (Supplementary Figure S3A). From a pool of randomly selected adaptors (#1-#30), eight were identified as petites (Supplementary Figure S3B). Both the progenitor BG2 strain and the non-petite TUN adaptors exhibited clear zones of inhibition (ZOI) with similar values for RAD and FoG. In contrast, the petite adaptors showed no detectable ZOI, indicating a high level of resistance to FLC (Supplementary Figures S4C,D).

From the experiments described above, we established a connection between respiratory deficiency and resistance to TUN, as well as altered susceptibility to FLC, characterized by decreased tolerance and increased resistance. Notably, these petites were selected either through exposure to TUN alone or a combination of TUN and FLC. To explore whether petites selected under different stress conditions exhibit similar phenotypes, we turned our attention to Ethidium Bromide (EtBr). EtBr is known to inhibit mitochondrial DNA (mtDNA) synthesis and induce degradation of pre-existing mtDNA, leading to the conversion of respiratory-sufficient yeast into respiratory-deficient petites (Goldring et al., 1970).

In our study, CG4 was cultured in YPD broth supplemented with EtBr. After 24 h of incubation, the culture was diluted and subsequently spread onto YPD plates. From this plating, six randomly selected colonies were tested for their ability to grow on YPG, which serves as an indicator of respiratory competency. None of the selected colonies could grow on YPG, confirming that all were indeed petites (Figure 4A). We designated these colonies as “EtBr-evolved petites.”

Further analysis using a spot assay revealed that the EtBr-evolved petites exhibited superior growth compared to the progenitor strain in the presence of 8 μg/mL TUN, indicating a level of resistance to TUN (Figure 4B). Additionally, results from a disk diffusion assay demonstrated that all six petites had clear but smaller ZOI in comparison to the progenitor strain, suggesting that they lost FLC tolerance while gaining FLC resistance (Figure 4C).

Resistance to azoles typically arises from increased efflux and alterations in the target (Lee et al., 2023). In the C. glabrata genome, drug efflux is primarily mediated by ATP-binding cassette transporters, particularly through the C. glabrata sensitivity to 4-Nitroquinoline N-oxide (CgSNQ2) and C. glabrata Drug Resistance 1 and 2 (CgCDR1 and CgCDR2) genes (Hassan et al., 2021). Moreover, PDR1 encodes the central transcription factor that triggers the expression of CDR1 (Moye-Rowley, 2020). Brun et al. identified that resistance in FLC-induced C. glabrata petites was attributed to the upregulation of efflux genes, particularly CDR1 (Brun et al., 2004). In Saccharomyces cerevisiae, a model yeast closely related to C. glabrata, various respiratory inhibitors have diverse impacts on ergosterol biosynthesis (Adams and Parks, 1969). We hypothesize that TUN-induced and EtBr-induced petites might influence the expression of efflux and/or ERG genes, thereby enhancing FLC resistance while reducing FLC tolerance. Consequently, we compared the gene expressions between two petites and the parent CG4. One petite was induced by TUN exposure (TUN-induced petite, TiP), while the other was induced by EtBr exposure (EtBr-induced petite, EiP). Our findings revealed a significant increase in the expression of CDR1 and PDR1 compared to CG4 (p < 0.001, two-tailed Student’s t-test), whereas the expression of most ERG genes, including ERG1, ERG2, ERG3, ERG6, ERG7, ERG9, ERG11, ERG24, ERG25, was significantly reduced (p < 0.001, two-tailed Student’s t-test) in both types of petites (Figure 5A).

Next, we investigated the effect of TUN exposure on the expression of efflux and ERG genes. Exposure of CG4 to 8 μg/mL of TUN significantly down-regulated multiple ERG genes, including ERG1, ERG2, ERG3, ERG9, ERG11, and ERG24 (p < 0.001, two-tailed Student’s t-test). However, TUN had negligible effects on the expression of CDR1, CDR2, SNQ2, and PDR1 (p > 0.05, two-tailed Student’s t-test) (Figure 5B).