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. 2024 Aug 16;65(2):1217–1224. doi: 10.1007/s12088-024-01376-6

In Vitro Fluconazole Exposure Promotes Switch Phenotypes With Altered Virulence Traits in the Pathogenic Yeast Candida Tropicalis

Murilo Moreira dos Santos 1,2, Cássia Milena de Souza 1,2, Luciana Furlaneto-Maia 1,2, Marcia Cristina Furlaneto 1,2,✉
PMCID: PMC12246352  PMID: 40655387

Abstract

Candida tropicalis is an opportunistic yeast pathogen that has the ability to develop resistance to fluconazole. This study evaluated the effect of fluconazole on the rise of phenotypic switching in C. tropicalis, an event that affects virulence in this species. C. tropicalis clinical isolate 49.07 switched reversibly at high frequency (10−1 to 10−2) after growth in presence of subinhibitory concentration of fluconazole (0.0625 µg/mL). Phenotypic switching resulted in colonies exhibiting Star and Micro morphologies. A decrease in sensitivity to fluconazole for the Star morphotype was observed. Colonies of both switched morphotypes were associated with higher percentages of filamentous growth relative to the Smooth morphotype (clinical isolate pattern). Under filament-inducing conditions, filamentation was increased for the Star morphotype. The Star and Micro phenotypes exhibited reduced biofilm compared to the Smooth morphotype. The expression of transcription factors (EFG1 and WOR1) was increased in the Micro morphotype compared to the Smooth colonies. Our findings indicate that in vitro exposure of C. tropicalis to fluconazole may promote phenotypic switching and the occurrence of switched morphotypes with altered virulence traits.

Keywords: Switch states, Fluconazole, Filamentous growth

Introduction

Candida infections are a growing threat to human health. Candida tropicalis is one of the most commonly isolated Candida species in clinical settings worldwide [1], being particularly common in tropical regions [2–6]. For instance, C. tropicalis is the most frequently encountered Candida species in India [5, 7]. In Brazil, C. tropicalis is the second or third most isolated species [2, 8].

Mortality associated with C. tropicalis candidaemia can be higher than that associated with other non-Candida albicans species [9]. Importantly, the emergence of fluconazole-resistant C. tropicalis isolates in clinical settings has been reported [1, 4, 7, 10–13], with the highest rates in the Asia region [1, 11, 12].

C. tropicalis plays an active role in the infectious process through the action of various virulence factors, including filamentous growth, secretion of lytic enzymes, adhesion and biofilm formation, which intensify the epidemiological scenario [14]. An important feature of C. tropicalis is its ability to exhibit multiple forms of reversible phenotypic switching reflected by an assortment of variant colony morphologies [15, 16]. Soll et al. [15] described for the first time that C. tropicalis can undergo a switch during the course of an infection. Our group has demonstrated a naturally occurring in vitro switching between a number of phenotypes in clinical isolates of C. tropicalis [16]. A study of Souza et al. [17] showed that the structure of switch colony morphotypes of C. tropicalis is associated with the arrangements of extracellular matrix and filamentous growth. According to these authors, some switch colonies exhibited increased expression of regulators of the white-opaque switch transition (EFG1 and WOR1) in C. tropicalis [17].

Phenotypic switching in C. tropicalis has been associated with marked virulence traits, including filamentation, adhesion and biofilm formation capabilities as well as altered antifungal sensitivity [16, 18–21]. Virulence towards larvae of Galleria mellonella was also affected by switching in C. tropicalis [22]. Thus, switched strains of C. tropicalis may represent a problem in the clinical context due to the randomness of the event that can raise strains with higher virulence than the original clinical strain. Considering that phenotypic switching occurs in response to environmental cues and that fluconazole is a commonly used azole class of ergosterol biosynthesis inhibitors, especially in developing countries, we investigated the influence of fluconazole in promoting phenotypic switching in C. tropicalis. To this end, we assessed the increase in switched morphotypes following growth in the presence of fluconazole. Furthermore, we assessed the minimum inhibitory concentration for fluconazole and virulence traits in switched strains.

Material and Methods

Isolate of C. tropicalis

Isolate 49.07 of C. tropicalis isolate was recovered from the tracheal secretion from a patient of University Hospital of Londrina – Parana State, Brazil [23]. The smooth colonies of 49.07 are round with a smooth dome surface and smooth edges [16]. The fungal was stored as frozen stocks with 15% glycerol at -80ºC and cultured on yeast extract-peptone-dextrose (YPD) (DIFCO) agar plates (1% yeast extract, 2% peptone, 2% dextrose and 1.5% agar) at 30 °C for 96 h, for fungal reactivation.

Fluconazole Exposure

The method of fluconazole exposure was performed as described by Barchiesi et al. [24], with modifications. The Minimum Inhibitory Concentration for fluconazole (Sigma-Aldrich, USA) was previously determined by the broth microdilution test considering the parameters established by EUCAST (Edef 7.3.2, 2020) [25]. Thus, the subinhibitory concentration was defined as MIC/2. To this end, a cell suspension of 1 × 106 yeasts/mL in RPMI-1640 (Sigma-Aldrich, USA) medium was inoculated into fresh RPMI-1640 medium supplemented with the subinhibitory concentration of fluconazole (MIC/2 = 0.0625 µg/mL). The exposure lasted for 10 days, under incubation at 35ºC/100 rpm. Every 24 h aliquots containing 1 × 106 yeasts/ml were transferred into a fresh medium containing the same fluconazole concentration and reincubated. At the end of the 10th day, 1 mL aliquot of the suspension was inoculated onto YPD agar. As a control, the same growth procedure was done without the presence of fluconazole.

Selection of Morphological Patterns, Frequency, and Reversibility

After 10th-day fluconazole exposure, colonies exhibiting the smooth phenotype (isolate phenotype) and displaying distinct morphological patterns (morphotypes) were photographed using a stereoscopic microscope (Nikon SMZ-745) and scored. A total of the 4500 colony forming units (CFUs) were counted and the frequency was determined.

The reversibility of colony types displaying distinct morphology to the smooth phenotype (parental phenotype) was determined by visually scoring four days old-growth colonies on YPD agar medium (Gibco) at 28 °C. To calculate the reversibility frequencies, a total of 4500 colonies were scored. Colonies that arose from colony types displaying distinct morphology and exhibited smooth patterns (isolate-like phenotype) were considered revertant colonies.

Evaluation of the Minimum Inhibitory Concentration of Fluconazole

Cells of typical colonies of the post-exposure morphotypes were suspended in RPMI 1640 medium (1 × 105 yeasts/mL). Subsequently, 100μL of the suspension was inoculated into the microtiter plate together with 100μL of medium with antifungal, in concentrations from 0.03125 μg/mL to 16 μg/mL, resulting in a final volume of 200μL per well. Assays were incubated at 35ºC at 100 rpm for 24 h and optical density (O.D) was measured by spectrophotometer at 490 nm. MIC was defined as the lowest concentration of antifungal capable of promoting 50% growth inhibition (MIC50). Data were interpreted according to Edef 7.3.2 (EUCAST) and expressed as sensitive (S ≤ 2 μg/mL) (EUCAST, 2020) [25].

Quantification of Filamentous Forms in Colonies with Different Phenotypes

Cells from colonies of each morphotype were examined microscopically after 96 h of incubation to evaluate differences in their morphology, especially concerning differentiation into filamentous forms as described elsewhere [16]. To estimate the number of filamentous forms, 3000 cells of each morphological group were directly counted using a Neubauer counting chamber. Three colonies for each morphological group were analyzed. Filamentous forms were interpreted as hyphae and pseudohyphae. Calcofluor white M2R (10 μg/mL) (Sigma-Aldrich, USA) staining was used to show the filamentous forms qualitatively. The representative images were obtained using an epifluorescence microscope (Zeiss, Germany).

Filamentous Growth in Liquid Cultures

The filamentation ability was determined microscopically according to Galan-Ladero et al. [26], with some modifications. Briefly, cells of morphotypes colonies were incubated into yeast extract peptone-dextrose (YPD) broth and incubated at 120 rpm at 30 °C overnight. These cultures were harvested by centrifugation at 5000 × g for 5 min, washed twice with PBS and used to inoculate in non-inducing medium (YPD, 30 °C) and inducing medium (0.67% yeast nitrogen base + 0.75% glucose + 10% fetal bovine serum, 37 °C) [27]. The cultures were shaken at 200 rpm for 4 h. Finally, 1000 cells of each sample was observed at 40 × magnification to evaluate the hyphal morphology under an inverted light microscopy (Nikon Eclipse Ti-S). All experiments were repeated at least three times for each morphotype.

Adhesion and Biofilm Formation

The adhesion and biofilm formation assays of C. tropicalis morphotypes on polystyrene were performed according to the methodology previously described by Souza et al. [21], with modifications. Standardized cell suspensions from strain (200 µL) containing 1 × 107 cells/mL) were distributed in 96-well microplates and incubated at 37 °C for 1 h for adhesion, and 48 h for biofilm, without agitation. For the biofilm, after 24 h, 100 µL of the suspension was removed, and an equal volume of fresh medium was added to complete the 48 h of incubation. Adhesion and biofilm formation on polystyrene was determined by total biomass using the crystal violet (CV) staining method. Assays were performed in triplicate in three independent replications.

Relative Gene Expression

WOR1 (White-opaque regulator 1) and EFG1 (Enhanced filamentous growth protein 1) expression was determined by quantitative PCR (qPCR) after 48 h of morphotypes growth on solidified YPD. Cells of colonies with typical morphology were frozen in liquid nitrogen, and their RNA was extracted using the Trizol-chloroform method (Thermo Fisher Scientific, USA). RNA was quantified and the quality was assessed using a NanoDrop spectrophotometer (ThermoScientific, Loughborough, UK). Complementary DNA (cDNA) was synthesized from 200 ng of extracted RNA using an RT-PCR kit (Invitrogen, Carlsbad, CA, USA) in a GeneAmp® PCR (Eppendorf, Gradient Mastercycler) following the manufacturer's instructions. The cDNA obtained was stored in a freezer at − 20 °C.

For qPCR the following primers were used: WOR1—forward 5′-CCGTCTAATGTTATACCTGCATCAA-3′ and reverse 5′- TTCGTCGTACTTATGGTAATTATTGTTTTCT-3′ [28]; EFG1—forward 5- TTCAACTGCTGGACAACCAC-3′ and reverse 5′- TACCAGGAGGTTGGAATTGG-3′ [17]; β-actin (ACT1)—forward 5′-GGGACGATATGGAGAAGATCTG-3′ and reverse, 5′-CACGCTCTCTGTGAG GATCTTC-3′ [17]. Each sample was analyzed in duplicate by real-time PCR performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems®). The reaction was performed using Platinum® SYBR® Green qPCR Supermix-UDG (Invitrogen, Carlsbad, CA, USA) with a final volume of 20 μL. Gene expression data were normalized by the expression of the constitutive β-Actin gene (ACT1). Once the data were normalized, the relative gene expression levels were analyzed by the Eq. 2−(Δ−Δct).

Statistics Methods

Each experiment was performed in triplicates on three different occasions. The statistical analyzes were performed using GraphPad Prism 8. Paired T-test was performed to analyze the differences among multiple means. A P-value of < 0.05 was considered statistically significant.

Results

Effect of Fluconazole Exposure on the Increase in C. tropicalis Switch Morphotypes

Exposure of the clinical isolate 49.07 to the subinhibitory concentration of fluconazole (0.0625 μg/mL) led to colonies with altered morphologies. In contrast, growth in the absence of fluconazole (control) resulted entirely in colonies with a smooth dome surface and smooth edges characteristic of the clinical isolate phenotype, as illustrated in Fig. 1A. Exposure to fluconazole resulted in three main morphological patterns: Smooth, Star, and Micro. The Smooth morphotype comprised colonies with the same morphology as the clinical isolate, with no visible change from preexposure to postexposure to fluconazole. The morphotype named Star exhibited an irregular edge and a more structured dome surface (structured colony). The morphotype named Micro exhibited a small-sized, irregular edge and dome surface. Of the 4500 colonies counted at the end of the 10th day of fluconazole exposure, a total of 2889 colonies exhibited the Micro morphotype (64.2%), 1530 colonies exhibited the Smooth morphotype (34%), and 81 colonies showed a Star morphotype (1.8%) (Fig. 1A). As shown in Fig. 1B, the emergence of structured colonies (morphotypes Star and Micro) from the clinical isolate (smooth phenotype) occurred at high frequencies.

Fig. 1.

Fig. 1

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Phenotypic switching induced by fluconazole exposure. A Images of Candida tropicalis colonies on YPD agar plates. Cells suspensions were obtained from growth in RPMI without fluconazole for 10 consecutive days (Control) and from the 10th day of consecutive exposure to fluconazole (RPMI + 0.0625 μg/ml). A1 Parental-type colony, A2 Smooth colony, A3 Star colony, A4 Micro colony. Colonies were photographed at 2 × power. B The frequencies of switching and reversibility between each phenotype are indicated. C Minimum inhibitory concentration (MIC50) to fluconazole of morphological groups

Considering that reversibility is a trait of a phenotypic switching event where switched morphotypes can revert to the original phenotype, testing was conducted for the switched Star and Micro morphotypes. We obtained spontaneously revertant strains from both switched morphotypes, as shown in Fig. 1B. The Micro morphotype exhibited a higher reversibility rate (1.14 × 10–1) than that observed for the Star morphotype (7.14 × 10–2). Reversibility rates were determined by dividing the number of colonies exhibiting a parental phenotype (smooth) by the total number of counted colonies (total of 4500).

In Vitro Susceptibility of Switched Morphotypes to Fluconazole

The MIC values for fluconazole were comparable for the clinical isolate (Smooth morphotype) and switched Micro morphotype cells (MIC50 = 0.125 µg/mL). For the Star morphotype, the MIC was lower (0.0625 µg/mL) than that observed for the Smooth morphotype (Fig. 1C).

Evaluation of Filamentous Growth in Switched Phenotypes

The percentage of filamentous forms (hyphae and pseudohyphae forms) in switched colonies was determined. Both switched morphotypes included a significantly higher percentage of filamentous forms than the Smooth morphotype. Of the 9000 cells directly counted for the Smooth morphotype, 0.29% were filamentous forms. For the Star and Micro morphotypes, 3.78% and 2.96% filamentation were observed, respectively (Fig. 2A).

Fig. 2.

Fig. 2

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Filamentous growth of Candida tropicalis morphotypes. A Filamentous forms within colonies cells. Figures show representative cells of morphotypes colonies. B Filamentous growth in liquid culture in the absence and presence of fetal bovine serum (FBS). The results are means of three independent experiments (1000 cells counted per experiment). Fluorescence microscopy images of Calcofluor White M2R stained cells, scale bar (10 µm). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001

Additionally, the capability of filamentous growth in liquid filament-inducing conditions was evaluated. The criteria for determining the level of filamentation was the percentage of cells in filamentous form. As shown in Fig. 2A, under standard non-filament-inducing condition (YEPD at 30 °C), the Star and Micro morphotypes showed higher filamentation than the Smooth morphotype. Under filament-inducing condition, including supplementation with 10% fetal bovine serum at 37 °C, filamentation was higher for the Star morphotype compared to that observed for the Smooth morphotype. In addition, for the Micro and Smooth morphotypes, a significant increase in the level of filamentation was observed under filament-inducing condition compared to that of standard condition (Fig. 2B).

Switch Morphotypes Showed Varied Adhesion and Biofilm Formation

As shown in Fig. 3, adhesion and biofilm formation on polystyrene surface were affected by the switching event. The Micro phenotype exhibited lower adhesion and biofilm compared to that observed for the Smooth morphotype. The Star phenotype also decreased the total biomass of biofilm, although no difference was observed for adhesion compared to the Smooth morphotype. In addition, the Star phenotype showed higher capabilities of adhesion and biofilm formation than the Micro phenotype.

Fig. 3.

Fig. 3

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Adhesion and biofilm formation of morphotypes of C.tropicalis obtained after fluconazole exposure (0.0625 µg/mL for 10 days). A adhesion and B biofilm were determined by total biomass (crystal violet assay—CV). Values are expressed as the means and standard deviation. (*) P < 0.05, (**) P < 0.01

Relative Gene Expression of WOR1 and EFG1 Transcription Factors

The Micro morphotype showed a significant increase in the expression of WOR1 and EFG1 (P < 0.05), important transcriptional regulators of switching compared to the Smooth colonies. Differently, no significant difference was observed in the level of gene expression between Star and Smooth morphotypes (Fig. 4).

Fig. 4.

Fig. 4

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Relative WOR1 and EFG1expression in 48 h colonies. Data were normalized with the housekeeping β-actin gene and relativized with the expression of the smooth phenotype. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Values are expressed as the means and standard deviation

Discussion

Invasive infection due to Candida species is recognized as a major cause of morbidity and mortality in the healthcare environment. The chemical class of azoles represents the largest family of anti-Candida drugs. The increasing global use of these drugs, in particular fluconazole, is one of the main causes of the dominant trend of infections caused by Candida non-Candida albicans species, including C. tropicalis [14, 29].

Although we have previously described a natural switching in C. tropicalis, the effect of fluconazole exposure on the occurrence of switched phenotypes is largely unexplored. In the present study, the occurrence of high frequencies of reversible switch morphotypes after fluconazole exposure, as shown in Fig. 1B, suggests that phenotypic switching in C. tropicalis may be promoted in the presence of this drug, as yeast cells from the control samples (not exposed to fluconazole) always produced colonies with the original phenotype. Similar results were obtained for Candida glabrata, where fluconazole-exposed yeast cells produced switched phenotypes with varying frequencies [30]. For the pathogenic yeast, Cryptococcus neoformans antifungal drug therapy promoted the emergence of switch variants in a murine infection model; however, phenotypic switching did not affect the antifungal susceptibility of the yeast [31]. According to these authors, the enhancement of a switch phenotype after drug therapy may be due to a mechanism for escaping drug action other than resistance to the drug. In the present study, the susceptibility assay provides evidence that switching may be correlated with changes in the MIC of fluconazole for the Star morphotype, in accordance with our previous findings on the susceptibility of switched strains [16]. Currently, little is known about the correlation between phenotypic switching and drug resistance in C. tropicalis.

Yeast-filamentous growth transition is important virulence factor for Candida species. In the present study, an interesting observation was the generation of switch morphotypes (Star and Micro) presenting higher amounts of filamentous forms in colonies compared to the Smooth morphotype, as well as higher levels of filamentation in liquid cultures. Further, our data showed distinct levels of filamentation under filament-inducing condition among morphotypes of C. tropicalis. Of note, the Star morphotype exhibited higher filamentation levels under both filament and not filament-inducing conditions compared to the Smooth and the switched Micro morphotypes (Fig. 2B). C. tropicalis is a polymorphic species exhibiting yeast, hyphae, and pseudohyphae cell types [14]. Hyphal growth seems to play a vital role in the virulence of C. tropicalis [32]. A correlation between C. tropicalis filamentation ability and the invasion and damage of epithelial cells has been reported [33]. In a previous study, we showed a correlation between adhesion ability to plastic and filamentation for switch morphotypes of C. tropicalis [21]. Thus, exposure of C. tropicalis to fluconazole may be associated with the emergence of switch morphotypes with altered characteristics related to yeast pathogenesis. According to Radford et al. [34], the relationship between C. albicans-switched colonies and microstructure helps to elucidate the relationship between in vitro colonial morphology and in vivo pathogenicity.

Finally, we analyzed the expression of WOR1 and EFG1, transcription regulators of the white-opaque switch transition in C. tropicalis [35, 36], by cells from colonies of morphotypes that emerged after exposure to fluconazole. Our data showed higher levels of both genes in the Micro than in the Smooth colonies (Fig. 4), that may be associated with increased amount of filamentous forms in colonies (10-times higher) compared to Smooth colonies. This data is in accordance to our previously findings where the expression of EFG1 and WOR1 correlated to the high capacity of a switch strain to form hyphae throughout colony development [17]. Differences between switched morphotypes (Star and Micro) suggest distinct gene expression profiles throughout their development.

Conclusion

The obtained results indicate the promotion of phenotypic switching events in C. tropicalis by exposure to subinhibitory concentrations of fluconazole. Additionally, the increase in switched morphotypes with a significant increase in filamentation capabilities compared to that observed for the clinical isolate and changes in fluconazole sensitivity can lead to important clinical implications.

Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. MMS and CMS were fellowship holder of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES). MCF is grateful to CNPq for the PQ fellowship.

Author Contributions

MMS and MCF conceived the research and designed the experiments, MMS and CMS carried out the experiments, MMS and MCF wrote-original draft, LF-M, MCF wrote de manuscript – review and editing. All authors read and approved the final manuscript.

Declarations

Conflict of interest

The Authors declared that they have no conflict of interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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