Parallel evolution of fluconazole resistance and tolerance in Candida glabrata

Abstract

Introduction: With the growing population of immunocompromised individuals, opportunistic fungal pathogens pose a global health threat. Candida species, particularly C. albicans and non-albicans Candida species such as C. glabrata, are the most prevalent pathogenic fungi. Azoles, especially fluconazole, are widely used therapeutic options.

Objective: This study investigates how C. glabrata adapts to fluconazole, with a focus on understanding the factors regulating fluconazole tolerance and its relationship to resistance.

Methods: This study compared the factors regulating fluconazole tolerance between C. albicans and C. glabrata. We analyzed the impact of temperature on fluconazole tolerance, and requirement of calcineurin and Hsp90 for maintenance of fluconazole tolerance. We isolated colonies from edge, inside and outside of inhibition zone in disk diffusion assays. And we exposed C. glabrata strain to high concentrations of fluconazole and investigated the mutants for development of fluconazole resistance and tolerance.

Results: We found temperature modulated tolerance in the opposite way in C. albicans strain YJB-T1891 and C. glabrata strain CG4. Calcineurin and Hsp90 were required for maintenance of fluconazole tolerance in both species. Colonies from inside and outside of inhibition zones did not exhibited mutated phenotype, but colonies isolated from edge of inhibition zone exhibited diverse phenotype changes. Moreover, we discovered that high concentrations (16-128 μg/mL) of fluconazole induce the simultaneous but parallel development of tolerance and resistance in C. glabrata, unlike the sole development of tolerance in C. albicans.

Conclusion: This study highlights that while tolerance to fluconazole is a common response in Candida species, the specific molecular mechanisms and evolutionary pathways that lead to this response vary between species. Our findings emphasize the importance of understanding the regulation of fluconazole tolerance in different Candida species to develop effective therapeutic strategies.

Keywords: Candida glabrata; Hsp90; antifungal resistance; antifungal tolerance; calcineurin; evolution trajectory.

Figure 1

Temperature-dependent fluconazole tolerance in C. glabrata strain CG4 (A) Disk diffusion assay: 100 μL of cells at a density of 1x10⁵ cells/mL were spread on YPD-agar plates, and 200 μg fluconazole was applied to the disks. (B) Spot assay: 3 μL of 10-fold serial diluted cells were spotted on YPD-agar plates containing various concentrations of fluconazole, as shown in the figure. Plates were incubated at 30°C or 37°C for 48 hours (A) or 48 hours (B), then photographed.

Figure 2

Comparative analysis of calcineurin and Hsp90 requirements for fluconazole tolerance in C. albicans and C. glabrata This figure compares the essentiality of calcineurin and Hsp90 for fluconazole tolerance in C. albicans and C. glabrata. Plates were supplemented with either calcineurin inhibitor cyclosporine A (0.5 μg/mL) or Hsp90 inhibitors radicicol (1 μg/mL), geldanamycin (10 μM) or NVP-HSP990 (10 μg/mL), as indicated in the figure. Fluconazole-containing disks (200 μg) were placed on the plates, which were then incubated at 30°C for 48 hours before being photographed.

Figure 3

Carbon source utilization and fluconazole tolerance of CG4 colonies from inside and outside of zone of inhibition (A) Disk diffusion assay showing the zone of inhibition (ZOI) of fluconazole of CG4. IZO (red arrow) and OZO (blue arrow) colonies were randomly selected for further analysis. The plate was incubated at 30°C for 24 h to allow for optimal growth of the colonies. (B) Growth of IZO and OZO colonies on YPD and YPG plates containing glycerol as the sole carbon source. Colonies were suspended in distilled water and adjusted to 1.0x10⁶ cells/mL before spotting on the plates. The plates were incubated at 30°C for 24 h, after which the colonies were photographed. (C) Disk diffusion assay showing the tolerance of IZO and OZO colonies to fluconazole. The disks contained 200 μg of fluconazole, and the plates were incubated at 30°C for 24 (h) For the parent, 10 individual colonies were tested as 10 biological replicates. The images were analyzed using diskImageR script to quantify RAD₂₀ and FoG₂₀ values.

Figure 4

Characterization of EZO colonies (A) DDA of CG4 at 37°C, showing colonies at the edge of ZOI, (EZO colonies, red arrow) with reduced growth. (B) Growth of 16 randomly selected EZO colonies on YPG medium. Colony #7 failed to grow on YPG, indicating petite mutant phenotype. (C) DDA results of all 16 EZO colonies, with RAD₂₀ and FoG₂₀ values quantified using the diskImageR script. Mean values of 3 biological replicates are shown. Disks contained 200 μg fluconazole. Plates were incubated at 37°C for 24h and then photographed.

Figure 5

Fluconazole selects diverse resistant and tolerant adaptors (A) Approximately one million cells of CG4 were spread on YPD plates containing 8-128 μg/mL fluconazole. The plates were incubated at 37°C for 3 days, then photographed. The red circle indicates the colonies chosen for further analysis. (B) From each plate containing 16-128 μg/mL fluconazole, 16 random colonies (adaptors) were chosen. All 56 adaptors were spotted on YPD and YPG plates, incubated at 37°C for 24 h, and then photographed. (C) All 56 adaptors were tested with DDAs using disks containing 200 μg fluconazole. The results are presented as follows: P indicates petites, NP indicates non-petites, R indicates resistance to fluconazole, and T indicates tolerance to fluconazole. RAD₂₀ and FoG₂₀ values were quantified using the diskImageR script, and the mean of 3 biological replicates is shown for each strain.

Figure 6

Requirement of calcineurin and Hsp90 for evolved resistance and tolerance Fluconazole-selected adaptors (P & R, NP & R, and NP & T) were tested with DDAs using YPD plates supplemented with calcineurin inhibitor cyclosporine A (1 μg/mL) or Hsp90 inhibitors radicicol (1 μg/mL), geldanamycin (10 μM) or NVP-HSP990 (10 μg/mL). The plates were incubated at 37°C for 48 hours and then photographed.