In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry

Abstract

The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of Candida albicans, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 microg ml(-1)) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 microg ml(-1)) and YO1-64 (MIC, 64 microg ml(-1)). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g., CDR1 and CDR2, and the azole target enzyme, ERG11. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values, P value) in the adapted strain (P value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in C. albicans.

FIG. 1.

Isogenicity and growth patterns of fluconazole-susceptible and -resistant C. albicans strains. (a) CARE-2 hybridization pattern of EcoRI-digested chromosomal DNA of the C. albicans adapted strains. The positions of the molecular size markers (in kilobases) are shown on the left-hand side of the figure. (b) Comparison of growth curve of the fluconazole-susceptible and -resistant strains: the cells were grown in YEPD media at 37°C. Aliquot of cells was taken every 2 h till 22 h, and the optical density was measured at A₆₀₀.

FIG. 2.

Susceptibilities of adapted strains to antifungal agents. The yeast cells were grown overnight on YEPD plates at 37°C. Cells were then suspended in normal saline to an optical density of 0.1 (A₆₀₀). Five microliters of fivefold serial dilutions of each yeast culture were spotted onto YEPD plates in the absence (control) and presence of the indicated drugs: fluconazole (8 μg ml⁻¹), ketoconazole (0.16 μg ml⁻¹), itraconazole (0.16 μg ml⁻¹), terbinafine (12.5 μg ml⁻¹), and nystatin (1 μg ml⁻¹). Growth differences were recorded following incubation of the plates for 48 h at 37°C. Growth was not affected by the presence of the solvents used for the drugs.

FIG. 3.

(a) Fluorescence polarization measurements in fluconazole-susceptible and -resistant strains. Fluorescence polarization measurements were carried out using fluorescent probe DPH, as described in Materials and Methods, at excitation and emission wavelengths of 360 and 426 nm, respectively. Each experiment was done in triplicate, and the values represent means ± standard deviations. (b) Percentage labeling of phosphatidylethanolamine (PE) in plasma membrane of C. albicans cells with fluorescamine. Cells were labeled with fluorescamine as described in Materials and Methods. After labeling, cells were washed and lipids were extracted. The percentage of derivatized PE was determined as described in Materials and Methods. The results shown are the means of more than six independent experiments ± standard deviations.

FIG. 4.

Northern blots of total RNA from fluconazole-susceptible and -resistant C. albicans strains. RNA was isolated from cells grown logarithmically and hybridized with probes specific for CDR1, CDR2, MDR1, and ERG11. Hybridizations were performed as described in Materials and Methods. The bottom panel shows the corresponding gel load (25S rRNA).

FIG. 5.

R6G uptake and glucose-induced R6G efflux from fluconazole-susceptible and -resistant strains. The assay was performed essentially as described in Materials and Methods (22). Cells from YO1-16 (⋄), YO1-32 (□), and YO1-64 (▵) were incubated with 10 μM R6G at 37°C. One mole of glucose was added after a 25-min incubation in glucose-free phosphate-buffered saline. The corresponding filled symbols represent the extracellular concentrations of R6G in the presence of glucose. The inset shows R6G efflux at 35 min post-glucose induction, which gives a comparison with controls incubated without glucose. Each bar indicates the standard deviations of mean of four sets of experiments.