Azole drugs are imported by facilitated diffusion in Candida albicans and other pathogenic fungi

Abstract

Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a K(m) of 0.64 μM and V(max) of 0.0056 pmol/min/10⁸ cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.

Figure 1. Effect of glucose on [³H]-FLC accumulation levels in C. albicans.

A. FLC accumulation in the presence of 2% glucose. B. FLC accumulation in the absence of glucose after cells were starved for 2 h. Strains  =  SC5314 (open squares), #1 (inverted triangles), #17 (triangles) DSY1050 (open circles), heat-killed SC5314 (filled squares). Samples were removed at 1, 2, 3, and 24 h. Samples were normalized to CPM/1×10⁸ cells (y axis) and compared to the incubation time (x axis). Error bars are included and most times are smaller than the symbol.

Figure 2. Kinetics of import of [³H]-FLC.

Import kinetics measure the initial accumulation rate (y axis) as a function of [³H]-FLC concentration (x axis), using C. albicans strain SC5314. The Michaelis-Menten equation was used to determine K_m of 0.64 uM and V_max of 0.0056 pmol/min/1×10⁸ cells. Results are a representative graph of a minimum of three independent experiments.

Figure 3. Imidazoles and triazoles compete for [³H]-FLC uptake, but 5FC and R6G do not.

Samples were grown and processed as outlined in Experimental Procedures. (A) Influx was tested in the presence of 10 x (grey bars) and 100 x (black bars) molar excess of unlabeled azoles, 5FC or R6G. Import of [³H]-FLC was measured at 24 h post incubation. Values are the average of three biological replicates with standard deviations, shown as a percentage of SC5314 import in the absence of unlabeled compounds. All azoles compete at a significant level at both 10X and 100X (p<0.05). (B) To quantify competition for [³H]-FLC import, increasing concentrations of unlabeled compound (KTC) were added to the incubation mixture and samples were analyzed for [³H]-FLC import at 10 m, 30 m, 60 m and 180 m. Using linear regression analyses, each square represents the rate of [³H]-FLC uptake in the presence of the unlabeled KTC. The kinetics are a measure of rate of import of [³H]-FLC (y axis) as a function of the log of unlabeled KTC concentration (x-axis). Dotted line graphically represents the calculation of IC_50.

Figure 4. [³H]-FLC import in S. cerevisiae mutants.

FLC import was measured at 24 h for strains S288C (wild type), gene deletion snf7, gene deletion doa4, and heat killed S288C. Import is expressed relative to import in the wild type strain. Data is average of three biological replicates. snf7, doa4, and heat killed cells are all significantly reduced compared to wild type (p<0.05).

Figure 5. R6G Efflux of S. cerevisiae mutants.

R6G efflux was measured over time for strains S288C (wild type, squares), gene deletion snf7 (triangles), gene deletion doa4 (inverted triangles), and heat killed S288C (circles). At each time point, 10,000 cells were measured for geometric mean fluorescence. Data is representative of three independent experiments. Data is expressed as fluorescence relative to the mean at t = 0. The relative mean fluorescence (y axis) is plotted against time in minutes (x axis).

Figure 6. [³H]-FLC import of FLC resistant clinical isolates.

Each circle represents the [³H]-FLC import of individual clinical isolates relative to the mean (y axis) at 24 h (x axis). Filled circles represent isolates that vary significantly from the mean (long horizontal line) plus standard deviations (shorter horizontal lines). Data is an average of four independent experiments.