Echinocandin-tolerant C. tropicalis contributes to caspofungin treatment failure

Among the 50 patients with C. tropicalis bloodstream infection, 36% (18/50) received prophylactic treatment with fluconazole or voriconazole (S1 Table). Furthermore, 60% (30/50) of these patients were neutropenic. The overall mortality rate was 40% (19/47) at 30 days from the onset of candidemia. Antifungal susceptibility testing on these isolates revealed that 46% (23/50) were resistant to both fluconazole and voriconazole, whereas all isolates demonstrated sensitivity to caspofungin and micafungin (S1 Table). Additionally, all isolates exhibited wild-type phenotype to amphotericin B, with geometric mean MIC value of 0.078 μg/mL for isavuconazole. Among the fluconazole-resistant isolates, 91% (21/23) harbored the Y132F and S154F double substitutions in Erg11, whereas one isolate did not exhibit any amino acid substitutions (S1 Table). Notably, caspofungin treatment failure (defined as persistent candidemia >10 days with positive blood cultures despite standard dosing: 70 mg loading dose followed by 50 mg/day maintenance) occurred in two patients infected with azole-resistant isolates SLT9 and SLT38 (fluconazole MIC = 128 μg/mL, S1 Table). Despite in vitro susceptibility to caspofungin (MIC ≤ 0.03 μg/mL) for these isolates, clinical resolution of the infections required a switch to amphotericin B therapy (S1 Table).

To evaluate echinocandin tolerance in clinical isolates, we employed a tolerance index (TI) to screen for echinocandin-tolerant strains, calculated as the survival fraction after 24-hour exposure to caspofungin or micafungin. Using a TI threshold >0.5, we identified eight tolerant isolates (Fig 1A and S1 Table). Notably, tolerant strains consistently displayed significantly higher TIs than non-tolerant strains for both caspofungin and micafungin (Fig 1B). Time-kill and dose-response analyses demonstrated persistent survival advantages in all tolerant isolates (SLT6, SLT9, SLT13, SLT21, SLT22, SLT34, SLT38, SLT44) relative to non-tolerant controls (SLT14, SLT2) and ATCC750 (Fig 1C). Tolerant isolates maintained stable survival rates across increasing caspofungin concentrations, in contrast to the dose-dependent viability decline in controls. This enhanced tolerance was further validated by spot growth assays comparing tolerant strains with ATCC750 (S1 Fig).

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Fig 1. The tolerance of Candida tropicalis isolates to echinocandins contributes to the therapeutic failure of caspofungin in vivo.

(A) Clinical isolates of C. tropicalis were cultured overnight, centrifuged, washed in PBS, and adjusted to a concentration of 2 × 10⁵ CFU/mL in YPD medium with 1μg/mL caspofungin or 1μg/mL micafungin. The cultures were then incubated at 37 °C for 24 hours. The tolerance index of each isolate was determined by comparing the CFU count to the initial inoculum. Isolates with a tolerance index exceeding 0.5 were classified as tolerant. (B) Statistical comparison of tolerance indices between the screened tolerant and non-tolerant strain groups under caspofungin (left panel) and micafungin (right panel) treatment was performed using the Mann-Whitney U test. The data are presented as the mean ± standard deviation (SD). (C) Both tolerant isolates and non-tolerant isolates, along with the reference strain ATCC750 were grown overnight in YPD medium, washed with PBS, and then inoculated at a density of 2 × 10⁵ CFU/mL into 2 mL YPD broth with or without caspofungin at 37 °C. Survival curves were constructed by normalizing the CFU counts of treated samples to those of untreated controls. Left panel represents the time course, while the right panel illustrates the dose-reponse relationship. (D) This survival analysis included 20 enrolled subjects with well-documented clinical data, who had experienced candidemia caused by a single pathogen and had received primary treatment with caspofungin for a minimum of one week without any evidence of concurrent infections from other pathogens. Subjects were categorized into "tolerance" and "non-tolerance" groups based on the echinocandin tolerance indices of the isolated strains as determined by the aforementioned results. Statistical comparison of tolerance indices between tolerance and non-tolerance groups following caspofungin treatment was performed using the Mann-Whitney U test (left panel). Survival data were analyzed using the Kaplan-Meier method, and statistical significance was assessed with the log-rank (Mantel-Cox) test (right panel). (E) Comparison of fungal burdens in Galleria mellonella larvae infected with tolerant strains and ATCC750, with or without caspofungin treatment. (n = 5 per group). Statistical analysis was performed using a two-way ANOVA. (F) Kaplan-Meier survival analysis of G. mellonella larvae (n = 20 per group) following infection with the tolerant strains or reference strain ATCC750 (inoculum: 1 × 10⁶ CFU/larva). Caspofungin-treated groups received 0.5 μg/larva at 1 h post-infection. Statistical analysis was performed using a log rank (Mantel-Cox) test. *, P < 0.05; ****, P < 0.0001; ns, not significant.

https://doi.org/10.1371/journal.ppat.1013220.g001

A retrospective cohort study was conducted to evaluate the clinical implications of in vitro caspofungin tolerance in 20 candidemia patients infected with C. tropicalis. Patients were stratified into tolerance and non-tolerance cohorts based on isolates' tolerance index. Strains from the tolerance cohort demonstrated significantly elevated TIs compared to non-tolerant counterparts (Fig 1D, left panel). Kaplan-Meier analysis revealed divergent 30-day survival outcomes (P = 0.011), with tolerance cohort patients demonstrating substantially lower survival rates (33%) compared to the non-tolerance cohort (85%) (Fig 1D, right panel). Notably, all surviving tolerance-group patients developed persistent C. tropicalis candidemia despite therapy, while none in the non-tolerance group showed recurrent infection (S1 Table).

To further validate that the in vitro observed strain tolerance constitutes a critical determinant of poor clinical therapeutic outcomes, we conducted systematic evaluations of both tolerant and non-tolerant strains using the Galleria mellonella larval infection model. Initial assessments demonstrated that larvae infected with tolerant strains SLT9 and SLT34 exhibited over 1,000-fold higher fungal burdens compared to those infected with the reference strain ATCC750 at 24h post-caspofungin treatment (Fig 1E). Survival analysis showed marked efficacy disparities: non-tolerant strains (ATCC750, SLT2, SLT14) exhibited 75–90% mortality reduction with caspofungin therapy, while six tolerant strains (SLT9, SLT34, SLT6, SLT21, SLT22, SLT44) showed no therapeutic response (Figs 1F and S2). Although caspofungin showed partial activity against tolerant strains SLT13 and SLT38 (15–20% mortality reduction), the effect remained substantially weaker than that observed against non-tolerant isolates (S2 Fig). These in vivo results conclusively demonstrate that the in vitro defined tolerance phenotype directly correlates with diminished therapeutic efficacy. We chose SLT34 and SLT9 for further mechanistic studies based on their contrasting phenotypes (highest vs. intermediate TIs, respectively) and clinical origins (breakthrough infection vs. persistent candidemia, respectively).

Adaptive morphological changes in caspofungin-tolerant strains during caspofungin exposure in vitro and in vivo

To further understand of the mechanisms by which echinocandin-tolerant isolates rapidly adapt and survive upon drug exposure, we initiated our investigation by employing population analysis profiling to screen for heteroresistance to caspofungin. No heteroresistance was detected, but we discovered atypical growth characteristics of the tolerant isolates on YPD plates containing caspofungin. Caspofungin-resistant strains produced creamy white, moist colonies on both drug-containing and drug-free plates. In contrast, all tolerant strains exhibited a delayed colony formation on the drug-containing plates, which occurred a day later, and the colonies were small, raised, and dry, composed of spherical cells and aggregated cells with abnormal divisions and enlarged size (Figs 2A and S3). Following caspofungin exposure in YPD medium, tolerant strains SLT9 and SLT34 developed multicellular aggregates due to incomplete cell division, retaining physical connections between mother-daughter cells (Fig 2B). These structures matured into three-dimensional grape-like clusters by 18 hours. Subsequent comprehensive analysis confirmed this aggregation phenotype in all remaining tolerant strains, demonstrating conserved stress adaptation (S4 Fig). In contrast, non-tolerant strains (ATCC750, SLT2 and SLT14) exhibited swollen, irregular, or even fragmented morphologies (Figs 2B and S4).

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Fig 2. Multicellular aggregation in tolerant strains following caspofungin exposure, as observed in vitro and in vivo.

(A) Cultures at the logarithmic growth phase of all strains were pelleted by centrifugation, rinsed with PBS, and then diluted before being spread onto YPD agar plates with and without 1ug/mL caspofungin. After a 48-hour incubation at 37 °C, images were captured. Subsequently, individual colonies were suspended in PBS, and stained with Calcofluor White (CFW) for microscopic analysis. ATCC750 is the reference strain, SHR001 is characterized as caspofungin-resistant, and SLT34 and SLT9 are classified as tolerant strains. The scale bar represents 20 µm. (B) Each strain was cultured in YPD liquid medium containing caspofungin, with an initial optical density (OD) of 2, and incubated at 37 °C with shaking. At 0, 6, and 18 hours' post-drug exposure, aliquots of the culture were taken, fixed with 4% paraformaldehyde, then stained with CFW, and observed under a fluorescence microscope. (C) The cell line RAW 264.7 was co-cultured with the strains at a multiplicity of infection (MOI) of 10, and various concentrations of caspofungin were added to the co-culture system. After an 18-hour incubation period at 37 °C, the samples were stained with CFW and then examined under a fluorescence microscope. (D) After a 48-hour infection of G. mellonella larvae with the strains, histopathological examination was performed. Tissue sections were stained using haematoxylineosin (HE) to assess general morphology and CFW to highlight specific cellular structures. White arrows indicate multicellular aggregate morphology. The scale bar represents 20 µm.

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To rule out the possibility that cell adhesion was causing the aggregation, we treated the aggregated cells with trypsin and proteinase K, which resulted in no change; however, ultrasonic treatment successfully dispersed the cells (S5 Fig). Following exposure to caspofungin, we performed PI (propidium iodide) staining on the yeast cells of the strains and observed that approximately 80% of the ATCC 750 strain cells exhibited signs of death after 6 hours of caspofungin exposure (S6 Fig). In contrast, the tolerant strain showed less than 40% cell death even after 18 hours of exposure to caspofungin. Moreover, the dead cells were predominantly found within the aggregated cellular clusters, while the dispersed cells remained largely viable.

We further co-cultured the tolerant strains with macrophages at an MOI of 10 and observed that even at low drug concentrations, the tolerant strains exhibited aggregation, which became more pronounced under higher drug concentrations (Fig 2C). After extending the culture period, we did not observe hyphal growth of the tolerant strains. However, when co-cultured with macrophages at an MOI of 1, hyphal growth was evident in the tolerant strains after 48 hours (S7 Fig). To explore whether this aggregative morphology exists in vivo, we constructed a Galleria mellonella larval infection model. We found that in untreated larvae, all strains caused extensive infection foci, with hyphal invasion leading to widespread tissue damage (Fig 2D). In larvae treated with caspofungin, the tissue pathology of the caspofungin-resistant strain SHR001 group showed no significant difference compared to the untreated group. In ATCC750 group, the infection foci were smaller, and the yeast cells were enlarged and spherical; in tolerant strains SLT9 and SLT34 groups, the infection foci were dispersed, and the yeast cells aggregated, and exhibited abnormal divisions (Fig 2D). This suggests that during the course of caspofungin therapy, tolerant strains could exhibit a multicellular aggregate morphology in vivo.

Given that the target of caspofungin is the glucan synthase enzyme in the fungal cell wall, we proceeded to further investigate the alterations in the cell wall of the tolerant strains under drug exposure. Scanning electron microscopy (SEM) revealed that under normal conditions, all strains exhibited an oval spherical cell morphology with smooth surfaces. Upon exposure to caspofungin, the ATCC750 cells were club-shaped, while the tolerant strains appeared as multicellular aggregates with wrinkled surfaces (Fig 3A). Transmission electron microscopy (TEM) more clearly confirmed that the multicellular morphology of the tolerant strains was caused by defects in cell division or budding processes (Fig 3B). Additionally, under caspofungin exposure, the ATCC750 cells contained an abundance of lipid droplets that had undergone fusion, with regions of the cytoplasm exhibiting dissolution. Furthermore, ultrastructural analysis revealed divergent cell wall remodeling strategies under caspofungin pressure. In the ATCC750 strain, we observed a significant 2.9-fold expansion in total cell wall thickness (from 179.6 to 526 nm; P < 0.001), accompanied by a 3.3-fold thickening of the inner cell wall layers (from 138.5 to 461.8 nm; P < 0.001) (Fig 3C). In contrast, the tolerant strains SLT9 and SLT34 exhibited only sporadic cell wall thickening in subpopulations of cells, with no statistically significant changes observed at the population level (Fig 3C). This phenotype divergence suggests tolerant strains may compensate through cell wall composition remodeling rather than bulk polysaccharide deposition.

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Fig 3. Changes in the cell wall of tolerant and non-tolerant strains under caspofungin treatment.

Morphological examination of tolerant and non-tolerant strains under scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (A-C). SEM images (A) and TEM images (B) are shown with scale bar as 1 µm. White arrows indicate intercellular septa. WCW (whole cell wall) and ICW (inner cell wall) indicate the measured regions of the cell wall structure. (C) Quantification of whole cell wall and inner cell wall thickness from TEM images. Cell wall measurements were performed using ImageJ with scale bar calibration. For each experimental group, five cells were randomly selected, and ten measurements were made from different sections of each cell. (D) Representative fluorescence images of the three main cell wall polysaccharide components in tolerant and non-tolerant strains under caspofungin stress at different time points. C. tropicalis cells were stained using ConA-FITC to delineate mannan, Fc-Dectin-1 to delineate β-glucan, and calcofluor white (CFW) to visualize chitin. The scale bar corresponds to 5 μm. (E) The monosaccharide composition of the cell wall in tolerant and non-tolerant strains under caspofungin stress, as determined by HPAEC-PAD. The values represent the percentage of each monosaccharide molecule, and these percentages are inferred to represent the polysaccharide components. The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using a two-way ANOVA. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013220.g003

The fungal cell wall is typically composed of mannans, glucans, and a minor fraction of chitin, with glucans and chitin constituting the primary components of the inner cell wall layer. We initially employed fluorescence staining to assess the variations in the composition of the cell walls of various strains. We observed that upon exposure to caspofungin, the ATCC750 strain exhibited a significant enhancement in the fluorescence signals of chitin and β-glucan over time, with a notable increase in the exposure of β-glucan (Fig 3D). In contrast, the tolerant strain showed a significant increase in chitin content, yet there was no apparent exposure of β-glucan (Fig 3D). This differential response may contribute to the tolerant strain's ability to evade immune recognition. Then the differences in polysaccharide constituents were detected using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). After a 6-hour exposure to caspofungin, an increase in chitin content within the cell walls was observed in all strains (Fig 3E). In contrast, following an 18-hour exposure, there was a significant rise (from 48.09% to 65.99%) in the relative proportion of glucan in the cell walls of the ATCC750 strain. Conversely, a significant reduction in glucan proportion was observed in the tolerant strains, with decreases from 45.15% to 36.59% for strain SLT34 and from 45.36% to 36.45% for strain SLT9 (Fig 3E). Notably, the HPAEC chromatograms of all strains were identical, with no extra peaks observed adjacent to the peaks corresponding to the three monosaccharides (S8 Fig), suggesting the absence of other monosaccharide derivatives.

Omics-based profiling unveils adaptive molecular regulation of cell wall and cell division in caspofungin-tolerant isolates

It was reported that mutations in non-hotspot regions of the FKS1 gene can lead to Candida parapsilosis' tolerance to caspofungin without conferring resistance [30]. Consequently, we sequenced the FKS1 gene of all tolerant isolates, but no missense mutations were identified (S1 Table). To explore whether the tolerance to caspofungin observed in the isolates identified in this study is associated with the paradoxical growth of strains under echinocandins, we examined the paradoxical growth of all isolates in the presence of caspofungin. We found that 37.5% of tolerant strains exhibited paradoxical growth, and 30% of the non-tolerant isolates showed paradoxical growth under caspofungin (S1 Table).

To further investigate the genetic basis of tolerance, we performed whole-genome sequencing on representative strains, including non-tolerant strains ATCC750, SLT14, and the tolerant isolates SLT9, SLT13, SLT21, SLT22, and SLT34. Comparative genomic analysis revealed substantial heterogeneity among these strains, and no conserved mutations directly linked to tolerance were identified (S9 Fig). However, GO and KEGG analyses of homozygous mutations shared by these tolerant strains showed enrichment in the categories of "Carbohydrate metabolism" and "Cell cycle" (S9 Fig). While these findings do not establish direct causality, they suggest that tolerance may emerge through polygenic adaptation involving metabolic and cell wall remodeling systems.

Subsequently, we selected the SLT34 strain and the control ATCC 750 strain for analysis using RNA-seq. Consistent with the substantial genomic variations, the mRNA expression profiles of the SLT34 strain and the ATCC70 strain also exhibit significant differences, with a total of 2,471 differentially expressed genes (DEGs) identified (Fig 4A and S2 Table). Under caspofungin stress, the ATCC750 strain shows a higher number of DEGs compared to the SLT34 strain (842 vs. 641), and the overlap of these DEGs is less than one-third (Fig 4B). Further GO enrichment analysis revealed that under caspofungin stress, genes upregulated in both the ATCC750 and SLT34 strains were significantly enriched in the GO terms "Cell wall organization or biogenesis" and "Chitin metabolic process" (Fig 4C). However, genes downregulated in the SLT34 strain were enriched in "Cell cycle", "Cellular response to stimulus", and "DNA replication", while those downregulated in the ATCC750 strain were enriched in "Carbohydrate metabolic process" and "Lipid catabolic process" (Fig 4C). Similarly, KEGG analysis indicated that under caspofungin stress, genes downregulated in the SLT34 strain were enriched in the "Cell cycle" and "DNA replication" pathways, whereas upregulated genes were enriched in the "MAPK signaling pathway" (Fig 4D). Analysis of the expression levels of genes related to cell wall polysaccharide metabolism and the cell cycle revealed that under normal conditions, the SLT34 strain exhibited significantly higher expression of cell wall-related genes, such as CHT3, MNN22, and GSC1, compared to the ATCC750 strain (Fig 4E). However, under caspofungin stress, the expression of these genes either decreased or showed no significant change. In contrast, the expression of cell cycle-related genes in the SLT34 strain, including ACE2, IQG1, and DBF2, significantly decreased under caspofungin stress (Fig 4E). Subsequently, we performed RT-qPCR analysis on a subset of genes associated with cell wall biosynthesis and the cell cycle, confirming that their expression patterns closely mirrored those observed in the RNA-seq data (Fig 4F). While the expression profiles of these genes in the tolerant strain SLT9 did not entirely match those of the SLT34 strain, the glucan synthesis gene GSC1 was consistently found to be highly expressed under normal conditions across these tolerant strains, with minimal changes in expression under caspofungin stress (Fig 4F). Additionally, the cell division-related genes ACE2 and IQG1 in these tolerant strains showed a significant decrease in expression in response to caspofungin stress. To explore the relationship between genetic variations in the tolerant strain SLT34 and the distinct expression patterns of corresponding genes under caspofungin stress, we integrated transcriptomic and whole-genome sequencing data. This integration identified several genes associated with mitosis, such as ENG1 and SCW11, which harbored homozygous indels (S3 Table). Notably, the expression levels of these genes exhibited a greater decrease in response to caspofungin stress compared to the ATCC 750 strain (S3 Table).

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Fig 4. Role of cell wall integrity and cell division pathways in C. tropicalis tolerance to caspofungin.

(A) Hierarchically clustered heatmap of RNA-seq expression profiles. ATCC750-T and SLT34-T represent strains treated with 1 μg/mL caspofungin for 1 h (n = 3 biological replicates per group). (B) A Venn diagram illustrating the differentially expressed genes following caspofungin treatment in the ATCC750 strain versus the SLT34 strain. (C) GO and (D) KEGG enrichment analyses are conducted on differentially expressed genes under caspofungin stress for both ATCC750 and SLT34 strains, where ATCC750-Up refers to the enrichment analysis outcomes for upregulated genes following caspofungin treatment in the ATCC750 strain, with other groups analyzed similarly. Selected terms and pathways are showcased. (E) Heatmap illustrating the expression levels of differentially expressed genes associated with cell wall integrity and cell cycle regulation across all groups. (F) mRNA levels of CHS1, CHS2, MNN22, MNN45, GSC1, CDA2, ACE2, and IQG1 genes are quantified by RT-qPCR, with data normalized to the expression of the ACT1 gene and presented relative to the untreated ATCC750 group. Data are expressed as mean ± SD and are representative of three biological replicates. Statistical analysis was performed using a two-way ANOVA with Tukey's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (G) Venn diagram comparing differentially expressed proteins between ATCC750 and SLT34 strains with or without caspofungin treatment, based on quantitative proteomic analysis. (H) GO enrichment analysis was performed separately on the differential expression proteins of the ATCC750 strain and the SLT34 strain after caspofungin treatment, with some GO terms displayed. (I) A table is presented showing some genes that have consistent expression patterns in both the transcriptome and proteome.

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Subsequently, proteomic analysis was performed and revealed significant baseline protein expression differences between SLT14 and ATCC750, with 667 differentially expressed proteins (DEPs) identified (S4 Table). Under caspofungin stress, the ATCC750 strain exhibited 572 DEPs, whereas the SLT34 strain showed only 373 DEPs, and the overlap of upregulated DEPs between the two was less than a quarter (Fig 4G). Further GO enrichment analysis revealed that these DEPs are predominantly enriched in GO terms related to "Carbohydrate catabolic process", "Regulation of response to stress", and "Glucose metabolic process" (Fig 4H and S4 Table). Next, we conducted a concordance analysis of the proteomic and transcriptomic data and found that under caspofungin stress, only 54 DEGs in the ATCC750 strain and 55 DEGs in the SLT34 strain exhibited consistent expression patterns at both the transcriptional and protein levels, with very few overlapping DEGs between the strains (S5 Table). Further analysis of these genes revealed that cell division-related genes ENG1 and SCW11 showed a good consistency between transcription and protein expression (Fig 4I).

Clinical isolates develop tolerance to caspofungin involving activation of chitin synthesis and downregulation of cell division-related genes

Based on our experimental results, it appears that the activation of chitin synthesis and the downregulation of cell division-related genes play a regulatory role in the tolerance of caspofungin by Candida tropicalis isolates. Nikkomycin Z, known for its inhibitory effect on chitin synthase enzymes, exhibits antifungal properties. We assessed the minimum inhibitory concentrations (MICs) of nikkomycin Z against ATCC750 and tolerant strains, revealing that the MICs consistently exceeded 16 μg/mL. However, the addition of nikkomycin Z significantly reduced the survival rates of tolerant strains under caspofungin, with a reduction in survival rates exceeding 10-fold (S10 Fig).

In order to further understand the mRNA expression characteristics of cell division-related genes identified by omics analysis in tolerant strains under caspofungin, we examined the mRNA expression of these genes at various time points after caspofungin treatment. We observed a significant reduction in the mRNA expression of the ACE2 gene in the tolerant strains following caspofungin treatment, particularly in the strain SLT34, where a nearly 100-fold decrease was noted after 4 hours (S11A Fig). This dramatic response was not observed in non-tolerant strains, which showed minimal changes in ACE2 gene mRNA levels post-treatment. Furthermore, in the tolerant strains SL9 and SLT34, the expression levels of both ENG1 and SCW11 genes plummeted by approximately 10-fold after caspofungin treatment (S11B and S11C Fig). This substantial decline in gene expression was maintained consistently over time. In contrast, the IQG1 gene in the tolerant strain SLT9 did not exhibit any significant alterations in its expression following caspofungin treatment (S11D Fig). Subsequently, we overexpressed Ace2, Eng1, and Scw11 individually in caspofungin-tolerant clinical isolates (SLT9 and SLT34) and assessed their survival rates under caspofungin. We observed that the overexpression of Ace2 in the tolerant strains SLT34 and SLT9 not only delayed the formation of multicellular aggregates under caspofungin, but also significantly reduced the survival rates of the tolerant strains post-treatment (Fig 5A and 5B). Specifically, the survival rates of strains SLT34 and SLT9 decreased by 100-fold and 20-fold, respectively. Additionally, the overexpression of Eng1 significantly reduced the survival rate of the SLT34 tolerant strain post-treatment, with a reduction magnitude of approximately 10-fold. However, overexpression of Scw11 did not exhibit these effects. Furthermore, the overexpression of Ace2 in strains SLT9 and SLT34 significantly improved the recovery rate of the infected larvae in a Galleria mellonella model (SLT9: 55% vs 25%; SLT34: 50% vs 15%) (Fig 5C).

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Fig 5. Overexpression of cell division-related genes partially restores caspofungin sensitivity in tolerant C. tropicalis strains.

(A) Overexpression of cell division-related gene ACE2 in tolerant strains significantly reduces fungal survival under caspofungin pressure. Following logarithmic phase growth, strains were adjusted to a density of 2 × 10⁵ CFU/mL in drug-supplemented YPD medium and incubated at 37 °C for 24 h. The yeast suspension was then diluted and 100 μL was spread onto YPD plates. After a 48-h incubation, CFUs were counted, and the survival rates were determined relative to the untreated control group. The data are presented as mean ± SD and are representative of three biological replicates. Statistical analysis was performed using a one-way ANOVA with Dunnett's test. ****, P < 0.0001. (B) ACE2 overexpression delays the development of caspofungin-induced multicellular aggregates in tolerant strains. Each strain was cultured in YPD liquid medium containing caspofungin, with an initial optical density (OD) of 2, and incubated at 37 °C with shaking. Aliquots of the culture were taken, fixed with 4% paraformaldehyde, stained with CFW, and observed under a fluorescence microscope at 0, 6, and 18 hours' post-drug exposure. (C) In Galleria mellonella infection models, ACE2 overexpression reverses therapeutic tolerance, as evidenced by improved larval survival following caspofungin treatment. Groups of larvae (n = 20 per group) were infected with 1 × 10⁶ CFUs of the respective strains and treated with or without caspofungin (0.5 μg/larva). Survival rates were assessed using Kaplan-Meier analysis, and statistical significance was determined using a log-rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01.

https://doi.org/10.1371/journal.ppat.1013220.g005

Calcineurin inhibitors can reverse the tolerance of C. tropicalis to caspofungin both in vitro and in vivo

In our quest to counteract the tolerance of C. tropicalis to caspofungin, we conducted a screening of various compounds to identify potential drugs that can reverse the tolerance. Fortunately, we have discovered that the calcineurin inhibitor FK506 is such potential drug. We observed that 1μg/mL of FK506 can completely inhibit the formation of multicellular aggregates by tolerant strains SLT9 and SLT34 exposed to caspofungin for 6 and 18 hours, respectively (Fig 6A). FK506 can suppressed the survival of all tolerant strains to caspofungin, exhibiting a concentration-dependent manner (S12 Fig). Upon the addition of 1 μg/mL FK506 to YPD plates containing caspofungin, it was found that the inclusion of FK506 could completely inhibit the formation of delayed growth small colonies by all tolerant strains under caspofungin stress (Figs 6B and S13A). Furthermore, the addition of FK506 markedly reduced the in vitro survival rate of all tolerant strains under caspofungin stress, by more than 10000-fold (Figs 6C and S13B). To determine whether the reversal of C. tropicalis tolerance to caspofungin is due to the inhibition of calcineurin or whether FK506 affects the tolerance through other pathways, we additionally conducted experiments to test the effect of another calcineurin inhibitor, cyclosporin A, in reversing the tolerance of C. tropicalis to caspofungin. Consistent with the effects observed following FK506 treatment, the treatment of cyclosporin A led to a reduction in the survival rate of tolerant strains under caspofungin stress, an effect that is concentration-dependent (S14A Fig). Specifically, a 10 μg/mL dose of cyclosporin A dramatically decreased the survival rate of the tolerant strains SLT9 and SLT34 by more than 10000 times under caspofungin. In addition, this concentration of cyclosporin A completely inhibited the formation of multicellular aggregate morphologies in strains SLT9 and SLT34 under caspofungin stress (S14B Fig).

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Fig 6. FK506 significantly potentiates caspofungin activity against tolerant C. tropicalis strains, reversing tolerance phenotypes in both in vitro and in vivo models.

(A) FK506 co-treatment completely inhibited multicellular aggregate formation in tolerant strains at both 6 h and 18 h post-caspofungin exposure. The strains were subjected to treatments with 1 μg/mL caspofungin or 1 μg/mL FK506, followed by washing and staining with calcofluor white (CFW). The samples were then examined and captured under a fluorescence microscope, with the scale bar indicating 20 μm. (B) FK506 completely prevents colony formation of tolerant strains on YPD agar plates supplemented with 1μg/mL caspofungin. Strains were cultivated to the logarithmic phase, harvested, and diluted to suitable concentrations before being spread onto caspofungin-containing YPD plates and incubated at 37 °C for 48 h, after which photographs were taken. (C) FK506 significantly suppresses the survival of tolerant strains in YPD liquid medium supplemented with 1 μg/mL caspofungin. Following logarithmic phase growth, strains were adjusted to a density of 2 × 10⁵ CFU/mL in drug-supplemented YPD medium and incubated at 37 °C for 24 h. The yeast suspension was then diluted and 100 μL was spread onto YPD plates. After a 48-h incubation, CFUs were counted, and the survival rates were determined relative to the untreated control group. (D) FK506 effectively reverses the alterations in cell wall polysaccharides induced by caspofungin across different strains. Post-treatment with caspofungin or FK506, the cell walls were isolated, hydrolyzed to monosaccharides, and analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to determine the monosaccharide composition, thereby inferring the polysaccharide composition of the cell wall in each strain. (E) FK506 counteracts the caspofungin-mediated adaptive gene expression changes in tolerant strains, as determined by RT-qPCR. The results were normalized to the expression levels of the gene ACT1 and are presented as relative values compared to the untreated ATCC750 strain. (F) FK506 substantially decreases the fungal load in G. mellonella larvae infected with tolerant strains and treated with caspofungin. Larval tissues were homogenized, appropriately diluted, and 100 μL was spread onto YPD plates containing antibiotics. After a 24-h incubation, CFUs were counted and the number of CFUs per larva was calculated. (G) Kaplan-Meier survival curves were constructed for G. mellonella larvae post-infection with the designated strains and subsequent treatment with the specified drugs. Groups of G. mellonella larvae (n = 20 per group) were infected with 1 × 10⁶ CFUs of the respective strains and treated with caspofungin or FK506 (0.5 μg/larva). Survival rates were assessed using Kaplan-Meier analysis, and statistical significance was determined using a log-rank (Mantel-Cox) test. Statistical comparisons in (C-F) were performed using two-way ANOVA with Tukey's post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013220.g006

Additionally, FK506 is capable of completely suppressing the increase in relative content of chitin and the decrease in relative content of glucan in the cell walls of tolerant strains exposed to caspofungin (Fig 6D). The RT-qPCR data demonstrate that caspofungin treatment significantly induces the expression of the chitin synthesis-related gene CHS2 across all tested strains. Conversely, FK506 notably reverses the caspofungin induced elevation of CHS2 mRNA levels in the ATCC750 strain and the tolerant strains SLT9 and SLT34 (Fig 6E). Furthermore, FK506 suppresses the elevation of mannan synthesis gene MNN22 mRNA levels in the SLT9 strain, the upregulation of glucan synthesis gene GSC1 mRNA levels in the ATCC750 strain, and the reduced expression of cell-division related gene ACE2 mRNA levels in tolerant strains SLT9 and SLT34 under caspofungin (Fig 6E).

Next, we assessed the therapeutic potential of calcineurin inhibition in reversing caspofungin tolerance using a Galleria mellonella infection model. For non-tolerant strain ATCC750, caspofungin monotherapy achieved near-complete fungal clearance, with FK506 co-administration showing no additive benefit. For tolerant strains, co-treatment with FK506 and caspofungin significantly reduced the fungal burden in larvae compared to caspofungin alone. Specifically, the number of colony-forming units (CFU) recovered from larvae infected with tolerant strains SLT9 and SLT34 was reduced by more than 100-fold (Fig 6F). Additionally, the survival rate of larvae infected with these tolerant strains was markedly improved when treated with the combined administration of FK506 and caspofungin, compared to treatment with caspofungin alone. Specifically, for larvae infected with strain SLT9, survival rates increased by 40%, while those infected with strain SLT34 exhibited a survival rate increase of over 60% (Fig 6G). This suggests that the combination therapy provides a substantial therapeutic advantage over caspofungin alone in managing infections caused by echinocandin-tolerant strains.

Ethics statement

The study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China (Permit Number: 2023-RE-346). Given the retrospective and observational nature of the study, the requirement for informed consent was waived.

Patients

Clinical data pertaining to patients diagnosed with C. tropicalis bloodstream infections were meticulously collected by accessing the electronic medical records database of the First Affiliated Hospital of University of Science and Technology of China from January 2017 and May 2023. Patients were included in this survival analysis if they met the following criteria: (a) they had at least one positive blood culture for C. tropicalis, (b) there was no concomitant isolation of bacteria or fungi from blood culture within two weeks from the first date of candidemia, (c) there was no evidence of concurrent infection with other pathogens within 48 hours of the first date of candidemia, and (d) they received primary treatment with caspofungin, which was administered for at least 7 days. The primary endpoint of this study was the assessment of 30-day survival rates.

Clinical isolate identification and growth

A collection of 50 clinical isolates of C. tropicalis was procured from 50 patients diagnosed with C. tropicalis bloodstream infections at the First Affiliated Hospital of the University of Science and Technology of China, spanning the period from January 2017 to May 2023. Blood samples for culture were collected from hospitalized patients, and yeast growth was identified using the BacT/Alert automated detection system (bioMérieux, France). The identity of each isolate as C. tropicalis was verified through Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS; Vitek MS, bioMérieux) or sequencing of the internal transcribed spacer (ITS) region. All isolates were grown on Sabouraud or YPD plates at 30 °C.

Antifungal susceptibility testing

The minimum inhibitory concentrations (MICs) of antifungal drugs were ascertained for 50 clinical isolates of C. tropicalis utilizing the broth microdilution technique, in strict accordance with the Clinical and Laboratory Standards Institute (CLSI) M27/4th edition guidelines. The antifungal agents assessed encompassed fluconazole, voriconazole, isavuconazole, micafungin, caspofungin, and amphotericin B, all procured from Selleck Chemicals (Houston, TX, USA). The MIC data for these drugs were interpreted in light of the CLSI documents M27M44S-Ed3 and M57S-Ed4. Concurrently, stringent quality control measures were implemented for each set of drugs, employing Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 as reference strains. In the detection of paradoxical growth, the concentration range of caspofungin was from 0.125 to 64 μg/mL, and paradoxical growth was defined as growth resurgence in at least two dilution wells above the MIC after 48 hours of incubation.

Echinocandins tolerance assessment

We initially screened for echinocandin tolerance by assessing survival following exposure to caspofungin and micafungin. In brief, all clinical isolates were cultured overnight in YPD medium, centrifuged, and washed with PBS. Fungal suspensions were standardized to 2 × 10⁵ CFU/mL in fresh YPD medium containing either 1 μg/mL caspofungin or micafungin. We selected this concentration because it represents the clinical resistance breakpoint for C. tropicalis. Cultures were then incubated at 37 °C with shaking at 220 rpm for 24 hours. Cells were subsequently harvested by centrifugation, washed three times with PBS, and plated on YPD agar for CFU counting. The tolerance index was calculated as the ratio of post-treatment CFU counts to the initial inoculum. Isolates exhibiting a tolerance index greater than 0.5 were categorized as tolerant. The selection of the cutoff for defining tolerant strains was based on the clear bimodal distribution observed across all clinical isolates, with minimal overlap between the tolerant and non-tolerant populations.

To validate the tolerance phenotypes identified in our initial screening, we performed standardized time-kill assays on all tolerant isolates along with the selected non-tolerant clinical isolates and the reference strain ATCC750. In brief, following overnight growth in YPD medium, cultures were washed with PBS and standardized to 2 × 10⁵ CFU/mL in fresh YPD broth with or without caspofungin supplementation. For time-dependent killing assessment, cultures were incubated at 37°C with agitation at 220 rpm, and aliquots were collected at 6, 12, 18, 24, and 48 hours' post-inoculation. The aliquots were serially diluted and plated on YPD agar for CFU counting. For dose-response assessment, cultures were treated for 24 hours under identical conditions. To ensure accurate quantification of aggregated cells, samples were sonicated for 50 s using an ultrasonic homogenizer (HD 2070.2, WIGGENS, Germany) prior to serial dilution and plating. Survival percentages were calculated relative to untreated controls at each point. All experiments included three biological replicates.

Galleria mellonella model for in vivo therapeutic experimentation

The Galleria mellonella infection model was established following established protocols for studying C. tropicalis pathogenesis and antifungal drug efficacy [41]. G. mellonella larvae, exhibiting a length of 2.5 to 3.0 cm and a weight of 0.25 to 0.3 g, were procured from a commercial supplier (Keyun Biotechnology Company, China). Upon acquisition, the larvae were subjected to a stringent health assessment. Subsequently, the larvae were randomly assigned into multiple experimental groups, with 20 larvae in each group. Larvae were inoculated via the last left proleg with 10 μL of C. tropicalis suspension (1 × 10⁶ CFU/larva), a site chosen for its direct access to the insect's hemocoel, an open circulatory system where hemolymph bathes all internal organs. This injection method delivers fungal cells directly into the hemolymph, the functional equivalent of mammalian blood, enabling whole-body dissemination post-infection [42]. For the antifungal therapeutic protocol, the larvae were injected into the last right proleg with caspofungin (0.5 μg/larva) after 1 hour of infection. The uninfected larvae (Mock group) were used as a control group that received an equivalent volume of the drug. The larvae were incubated at 37 °C, with daily survival assessments conducted over a 14-day period. These experiments were meticulously repeated on at least two occasions.

Furthermore, the fungal burden within the larval tissue was assessed 24 hours' post-infection, with five larvae per group. Specifically, individual larvae were subjected to homogenization using a FastPrep-24 5G apparatus (MP Biomedicals, Shanghai, China) with a speed of 6.0 m/s for 40 sec. The homogenates were serially diluted and aliquots were spread onto YPD agar plates. Subsequently, the plates were incubated at 35°C for 24 hours. After incubation, the colonies were counted to determine the number of colony-forming units per larva (CFU/larva). The fungal burden in the larvae was then expressed as CFU/larva. For histopathological examination, three larvae of each group were fixed in a 4% formaldehyde solution for 30 days and the histological sections were stained using haematoxylineosin (HE) and Calcofluor white (CFW).

Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) analysis

Yeast cells were collected and fixed in a solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4 °C for 2 hours to maintain cell integrity. Post-fixation, the samples underwent a dehydration process using an ethanol gradient (30% to 100%). Dehydrated samples were then subjected to critical point drying to eliminate the ethanol and any associated water content. Following this, the samples were sputter-coated with a thin layer of gold-palladium to enhance conductivity and image quality. The sputter-coated samples were examined using a scanning electron microscope (Hitachi, Japan) operated at an accelerating voltage of 10–20 kV. SEM images were obtained at various magnifications to assess the surface morphology of the yeast cells.

For TEM analysis, yeast cells were postfixed with 1% osmium tetroxide, dehydrated through an ethanol series, and infiltrated with an epoxy resin. Ultrathin sections were cut using an ultramicrotome (Leica, Germany) and collected onto copper grids. The ultrathin sections were stained with uranyl acetate and lead citrate to increase contrast and visualize cellular details. The stained sections were then examined under a transmission electron microscope at an accelerating voltage of 80–100 kV. TEM images were captured to elucidate the ultrastructure of the yeast cells. The measurement of cell wall thickness was conducted as described previously [43]. In brief, the cell wall thickness was systematically measured from TEM images using ImageJ with scale bar calibration. For each experimental group, five cells were randomly selected, and ten measurements were made from different sections of each cell.

Cell wall composition analysis

The cell walls were extracted following a previously described protocol [43]. Briefly, strains were inoculated and grown overnight, then diluted 1:100 into fresh medium. Subsequently, the cultures were exposed to either PBS or 1 μg/mL caspofungin for 6 and 18 hours, respectively, at 37 °C with constant shaking. The yeast cells were harvested, washed, and resuspended in sterile water before being lysed using a FastPrep-24 5G cell disrupter apparatus (MP Biomedicals, Shanghai, China). The resulting pellet was washed with 1 M NaCl and then treated with a protein removal buffer by boiling for 10 minutes. After this, the cell wall was rinsed with water and subjected to freeze-drying using liquid nitrogen. Accurate weighing of 5 mg of the lyophilized cell wall powder was carried out. Following hydrolysis to monosaccharides, the samples were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS 5000 system (Thermo Scientific, American). The monosaccharide composition of the cell wall polysaccharides, including glucan, chitin, and mannan, was quantified and the respective polysaccharide content within the cell wall was determined.

Fluorescence staining and confocal microscopy imaging of cell wall polysaccharides

The staining of cell wall polysaccharides was conducted according to a previously established protocol [43]. The strains were cultured overnight and then transferred to fresh medium for further incubation. Subsequently, the cultures were treated with either PBS or 1 μg/mL caspofungin for 6 and 18 hours, respectively. The cells were harvested and washed with PBS. And then the cells were fixed in 4% paraformaldehyde overnight at 4 °C. For mannan labeling, the fixed cells were washed with PBS and stained with Concanavalin A (ConA; Thermo Fisher Scientific, C11252) for 1 hour at 37 °C in the dark. β-glucan labeling involved a post-fixation wash with PBS, followed by staining with Fc-hDectin-1a (InvivoGen, fc-hdec1a) at 4 °C overnight. An Alexa Fluor 555-conjugated anti-human IgG antibody (Invitrogen, A-21433) was utilized as the secondary antibody, applied to the primary-stained yeast cells for 1 hour at 37 °C in the dark. Chitin was identified using calcofluor white (CFW; 30 μg/mL) with a 10-minute incubation at 37 °C in the dark. After staining, the cells were centrifuged and washed three times in PBS. The stained cells were then visualized with a confocal laser scanning microscope (Zeiss LSM800, Germany), and the images were captured and analyzed using ZEN Blue Lite software.

ERG11 and FKS1 sequencing

Genomic DNA was extracted from the target strains, and the ERG11 gene was amplified using the primers ERG11F (TTTGATTTATCACAGTTATAGACCC) and ERG11R (TGTATACTGTATTAAAGGCATAAATATG). Similarly, the FKS1 gene was amplified with the primers FKS1F (TTTATTTCAGGACACACCCACAC) and FKS1R (AACAAAGAACCAGTGGAAATGATTT). The purified PCR products were then sent to Tsingke Biotechnology Co., Ltd. (Nanjing, China) for bidirectional Sanger sequencing. The resulting sequences were aligned with the ERG11 reference sequence (GenBank: XM_002550939.1) and the FKS1 reference sequence (GenBank: EU676168.2) for comparative analysis.

Whole-genome sequencing (WGS)

The total genomic DNA of the strains was extracted and purified, followed by library preparation using Illumina's TruSeq DNA PCR-free prep kit according to the manufacturer's protocol. Library quality was evaluated with the Agilent High Sensitivity DNA Kit on an Agilent Bioanalyzer instrument. Libraries that met the quality criteria were then diluted in a gradient and mixed proportionally based on the desired sequencing depth. Following denaturation with NaOH to produce single-stranded DNA, the libraries were sequenced on the Illumina NovaSeq 6000 platform using paired-end sequencing.The raw sequencing data underwent quality control using fastp, an open-source tool designed to efficiently process and filter raw sequencing data to generate high-quality sequences. After quality filtering, the high-quality sequences were aligned to the reference genome (Candida tropicalis MYA-3404, GCF_000006335.3) using the bwamem algorithm with parameters set to default. SNP detection was conducted using the GATK software suite, while ANNOVAR was employed for annotating both SNP and InDel sites. Copy number variations (CNVs) across the genome were identified using the CNVnator software. The entire sequencing and analysis workflow was facilitated by Shanghai Personal Biotechnology Co., Ltd.

RNA-seq and bioinformatics analysis

The strains ATCC750 and SLT34 were incubated at 37 °C for 1 h, with and without the addition of caspofungin. RNA-seq analysis was performed as previously described [44]. In brief, the total RNA was extracted using a Quick-RNA Microprep kit (ZYMO Research, Seattle, USA) from both treated and untreated yeast cells. mRNA was purified from the input RNA material (3 μg) using poly T oligo-attached magnetic beads. The sequencing library was sequenced on the NovaSeq 6000 platform (Illumina, San Diego, USA) by Shanghai Personal Biotechnology Co., Ltd. Filtered reads were mapped to the reference genome MYA-3404 of C. tropicalis (GenBank: GCF_000006335.3). The uniquely mapped read counts were normalized, and the differentially expressed genes (DEGs) were identified using the fragments per kilobase of transcript per million mapped reads method with a false discovery rate adjusted to P < 0.05 and fold change values ≥ 2.0. Yeast samples from three independent biological replicates were analyzed.

Proteomic detection based on data independent acquisition (DIA) method

Strain ATCC750 and SLT34 were incubated at 37 °C for 1 h, with or without caspofungin treatment. After protein extraction from each sample, the BCA method was used for protein quantification, followed by trypsin digestion. The resulting peptides from the digested samples were desalted using a C18 Cartridge. An appropriate amount of iRT standard peptides was added to the tryptic peptides of each sample. DIA mass spectrometry analysis was conducted using a Orbitrap Astral high-resolution mass spectrometer (Thermo Fisher Scientific, USA). DIA data was processed with the DIA-NN software. In the screening for differentially expressed proteins between comparison groups, proteins with a fold change (FC) > 1.5 and a P value < 0.05 were selected. GO and KEGG annotations were applied to the target proteins using Blast2GO (version: BLASTP 2.8.0) and KOBAS (version: KOBAS 3.0) software, respectively. The workflow was supported by Shanghai Personal Biotechnology Co., Ltd. Yeast samples from three independent biological replicates were analyzed in this study.

Gene expression analysis using quantitative real-time PCR

Total RNA was extracted from yeast cells in the logarithmic growth phase using the Quick-RNA Microprep kit (ZYMO Research, USA). Complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit (TaKaRa,Tokyo, Japan). Then the quantitative PCR was performed using the SYBR Premix Ex Taq II (Tli RNaseH Plus) kit with the primers listed in S6 Table. The relative mRNA expression levels of the target genes were calculated using the 2^-ΔΔCt method, with ACT1 serving as the reference gene. Data for each gene were derived from three independent biological replicates.

Plasmid and strain construction

The construction of the gene overexpression strain was carried out through homologous recombination. In brief, we inserted the SAT1 cassette and the TDH3 promoter between the 1000 bp sequence upstream and 1000 bp sequence downstream of the target gene's start codon. The entire construct, complete with the necessary restriction enzyme sites at both ends, was synthesized by Tsingke Biotechnology Co., Ltd. using standard gene synthesis techniques. Following restriction enzyme digestion, the gene synthesis product was cloned into the vector pUC57. Subsequently, the ACE2 and SCW11 genes, after being digested with NdeI/NheI, were introduced into the SLT9 and SLT34 strains. Similarly, the ENG1 gene, digested with NheI/KpnI, was transformed into the same strains. Transformants resistant to nourseothricin were identified through colony PCR to verify that the TDH3 promoter had accurately replaced the native promoters of the target genes. Finally, RT-qPCR was used to assess the expression changes of the target gene mRNA in the mutant strains, and strains with the highest increase in gene expression were selected for further experiments. The primers, plasmids and strains used here are listed in S6 Table.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism version 10.3, with specific details provided in the corresponding figure legends. For comparisons involving more than three groups with two independent variables, a two-way analysis of variance (ANOVA) was employed, followed by post-hoc multiple comparison tests. Survival curves of Galleria mellonella were compared utilizing the Log-rank (Mantel-Cox) test. Data are presented as the mean or mean ± standard deviation (SD) for continuous variables. A P-value of less than 0.05 (two-tailed) was considered to indicate statistical significance.