Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans

Abstract

In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

FIG. 1.

Drug resistance profiles of C. albicans wild-type (WT) and erg mutant cells determined by a spot assay as described earlier (28). For this assay, 5-μl samples of fivefold serial dilutions of each yeast culture (each with cells suspended in normal saline to an OD₆₀₀ of 0.1) were spotted on YEPD plates in the absence (control) or in the presence of 4-nitroquinoline oxide (0.1 μg/ml), terbinafine (0.4 μg/ml), o-phenanthroline (4 μg/ml), fluconazole (0.4 μg/ml), itraconazole (0.1 μg/ml), and ketoconazole (0.02 μg/ml).

FIG. 2.

(A) Fluorescence imaging of C. albicans spheroplasts labeled with FAST-DiI. (a) Chemical structure of FAST-DiI. (b and c) C. albicans wild-type spheroplasts were labeled with 10 μM FAST-DiI (see Materials and Methods for details). Phase-contrast and fluorescence images of the same field of spheroplasts labeled in this manner are shown in panels b and c, respectively. (d to f) Confocal images of wild-type, erg2, and erg16 cells, respectively, labeled with 10 μM FAST-DiI. Confocal imaging was carried out by using an open pinhole under conditions described in Materials and Methods, and images were recorded on a 12-bit scale. Bars, 5 μm. (B) Fluorescence recovery plots after photobleaching of spheroplasts of C. albicans wild-type, erg2, and erg16 cells labeled with FAST-DiI. The difference in normalized fluorescence intensities between the prebleached cells and cells during recovery is plotted as μ over time. Fluorescence recovery profiles were analyzed as described earlier (17). Recovery was monitored for 120 s. The regression line drawn across the data points represents the rate of fluorescence recovery. A qualitative estimate of the diffusion coefficient can be obtained by comparing the slopes of the recovery plots. The plots indicate an increase in the diffusion coefficient for FAST-DiI in the order erg16 > erg2 > wild type. (C and D) Diffusion coefficients (C) and steady-state fluorescence polarization measurements (D) for C. albicans wild-type and erg mutant cells. Measurements were determined for spheroplasts by using DPH as the fluorescent probe and excitation and emission wavelengths of 360 and 426 nm, respectively, as described previously (28). The values are means and standard deviations (indicated by bars) of three independent experiments.

FIG. 3.

(A) Extracellular R6G concentrations in C. albicans wild-type (•), erg2 (▴), and erg16 (▪) cells at various time intervals. Deenergized C. albicans cells were incubated with R6G at 30°C. At various time points, cells were rapidly centrifuged, and the extracellular concentrations of R6G in the supernatants were determined spectrophotometrically at 527 nm. The values are the means and standard deviations (indicated by bars) of three independent experiments. (B) Accumulation of ³H-labeled fluconazole in deenergized C. albicans wild-type (•), erg2 (▴), and erg16 (▪) cells at various time intervals. (C) Measurements of steady-state fluorescence polarization of C. albicans wild-type cells in the absence (white bar) or in the presence of 12.5 mM (gray bar) and 25 mM (black bar) concentrations of BA. Steady-state fluorescence polarization was determined by using DPH as the probe as described in the legend to Fig. 2D. (D) Drug resistance profiles (microtiter assay) of C. albicans wild-type cells in the absence (white bar) or in the presence of 12.5 mM (gray bar) and 25 mM (black bar) concentrations of BA. The MIC₈₀ was determined as described earlier (28). Abbreviations: Flu, fluconazole; Keto, ketoconazole; Itra, itraconazole; Terb, terbinafine; Nqo, 4-nitroquinoline oxide; and Phe, o-phenanthroline.

FIG. 4.

Postlabeling transbilayer exchange of NBD-SM in C. albicans wild-type, erg2, and erg16 cells. Cells were grown in the absence (white bars) or in the presence (gray bars) of medium supplemented with 20 μg of ergosterol/ml. Cells were labeled with NBD-SM and then back extracted with 2% BSA as described in Results. The graph presents data for the 90-min time point, at which the maximum back-extracted fluorescence in the supernatant was observed. The values are the means and standard deviations (indicated by bars) of three independent experiments.

FIG. 5.

(A) Schematic representation of the yeast sphingolipid biosynthesis pathway. Fumonisin B1 inhibits the formation of phytoceramide from phytosphingosine. (B) Microtiter assays (MIC₈₀s) of C. albicans wild-type (WT) and erg mutant cells in the absence or in the presence of 50 μM fumonisin B1 (FumB1) were carried out essentially as described earlier (28).

FIG. 6.

Phase-contrast imaging (left panels) and fluorescence imaging (middle panels) under a confocal microscope of strain PSCDR1-GFP (A), strain PSCDR1-GFP grown in the presence of 50 μM fumonisin B1 (Fum B1) (B), and strain PSCDR1-GFP incubated with 5 μg of filipin/ml for 2 h (C). Cells were grown overnight and viewed directly for GFP fluorescence on a glass slide under a ×100 oil immersion objective in a Radiance 2100 (AGR3Q/BLD; Bio-Rad) confocal microscope equipped with 488-nm excitation and 500- to 530-nm band-pass emission filters. The fluorescence signal from strain PSCDR1-GFP showed the localization of Cdr1p on the plasma membrane. On treatment with Fum B1 or extended incubation with filipin, the GFP fluorescence from strain PSCDR1-GFP appeared to be concentrated inside the cells, indicating poor surface localization of Cdr1p. The right panels show the merge of the left and middle panels.

FIG. 7.

Glucose-induced R6G efflux from C. albicans cells. Deenergized cells were incubated with R6G for 2 h. Efflux was initiated by the addition of 2% glucose, and the concentration of the extruded dye in the supernatant was measured spectrophotometrically at 527 nm. R6G efflux is represented as the extracellular concentration of R6G in the supernatant 60 min after the addition of glucose to wild-type and erg mutant cells (A) and to wild-type cells with or without treatment with the indicated concentrations of fumonisin B1 (Fum B1) (B). The values are the means and standard deviations (indicated by bars) of three independent experiments.