Entry of ketoconazole into Candida albicans

Abstract

To define characteristics that determine the entry of ketoconazole (KTZ) into Candida albicans cells, we studied the uptake of [3H]KTZ. The cells rapidly and markedly concentrated the drug: 30% of the final 80-fold intracellular concentration was attained in less than 1 min, and >60% was attained in 10 min. Penetration of [3H]KTZ at an extracellular concentration higher than 0.1875 FM (0.1 ,ug/ml) occurred by a simple diffusion mechanism. At lower concentrations, accumulation of the drug was an active, energy-requiring process, dependent at least in part on glycolysis, and pH dependent (optimal pH, 6.6). The active transport system had a high binding affinity (Km = 50 nM) and a high maximum velocity of uptake (V..x = 1.4 ,umol min -10-7 cells). It was not possible to displace intracellular [3H]KTZ with high concentrations of unlabeled KTZ or other antifungal agents. These findings suggest that KTZ is rapidly taken up, highly concentrated, and tightly bound to cellular components of C. albicans.Ketoconazole (KTZ) (12) is a broad-spectrum antifungal agent which is active against a variety of pathogenic fungi, including Candida albicans and dermatophytes (5). Several investigators (13, 16) have shown that low concentrations of KTZ inhibit ergosterol synthesis by blocking C-14 demethylation. This fungistatic action can be related to the accumulation of a-methylated intermediates, such as lanosterol, in the fungal membrane. It can also be related to the depletion of ergosterol, which is specifically required for membrane integrity. A recent study (1) showed that high concentrations of KTZ can have a rapid fungicidal action by direct membrane-damaging effect on C. albicans cells.The high activity of KTZ in vivo against candidosis is in striking contrast to its low activity against C. albicans in vitro in conventional culture media (4).The ability of antifungal agents to enter yeast cells may be an important factor affecting pathogen susceptibility and the outcome of therapy.Because little is known about the factors which mediate the entry of KTZ into C. albicans cells and about the permeation process, we examined the in vitro uptake of this antifungal agent. MATERIALS AND METHODSOrganism and growth conditions. C. albicans IP 740, supplied by the Collection of the Institut Pasteur, Paris, France, was maintained on Casitone (Difco Laboratories, Detroit, Mich.) liquid medium at 37°C (5).Materials and drugs. Unlabeled KTZ and [3H]KTZ were gifts from Janssen Pharmaceutica, Beerse, Belgium. The labeled material has a specific activity of 445.8 mCi/mmol. Radioactivity was measured in a liquid scintillation counter (LKB 1211 Rackbeta liquid scintillation system).Determination of KTZ uptake by C. albicans. Cells grown in the absence of drug for 16 h were harvested, washed three times, and incubated in 0.1 M phosphate buffer (pH 7.4) at 370C.For most experiments, [3H]KTZ was added to a suspension of yeast cells at a final concentration of 0.1875 ,uM (0.1 ,ug/ml; containing 83.9 nCi of radioactivity per ml), which is the MIC for...

Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans

“…On the other hand, metabolic inhibitors were added to C. albicans cells to evaluate the active transport system. Sodium azide was used an inhibitor of oxidized cytochrome oxidase and DNP as an uncoupler of oxidative phosphorylation (Boiron et al 1987;Viejo-D ıaz et al 2004). Uptake of ZnPPc 4þ was little affected by the presence of azide and DNP mainly when the C. albicans cells were preincubated with these compounds for 60 before treatment with the phthalocyanine (Fig 5).…”

“…Therefore, the amount of ZnPPc 4þ bound to C. albicans cells was examined under different conditions, such as photosensitizer concentrations, incubation times, washing steps, cellular densities, and temperatures. Also, the uptake was evaluated in presence metabolic inhibitors (Boiron et al 1987;Bliss et al 2004). Taking into account these conditions of uptake, the photodynamic action of ZnPPc 4þ was examined to inactivate C. albicans cells.…”

Inactivation of the FCY2 Gene Encoding Purine-Cytosine Permease Promotes Cross-Resistance to Flucytosine and Fluconazole in Candida lusitaniae

“…To date, very little is known about the way in which antifungal agents enter the cell. To our knowledge, only one study measured the uptake of [ 3 H]ketoconazole and concluded that an energy-dependent uptake system was present for low concentrations of ketoconazole in C. albicans (4). Moreover, it has recently been shown that the uptake of the echinocandin drug caspofungin is mediated by a high-VOL.…”

Fluconazole resistance due to energy-dependent drug efflux in Candida glabrata

“…The intracellular volumes of the sus-ceptible and resistant cells were estimated by measuring 3 H 2 O uptake by the method described by Boiron et al (4).…”