Detection of inhibitors of Candida albicans Cdr transporters using a diS-C3(3) fluorescence

Abstract

Candida albicans is a major cause of opportunistic and life-threatening, systemic fungal infections. Hence new antifungal agents, as well as new methods to treat fungal infections, are still needed. The application of inhibitors of drug-efflux pumps may increase the susceptibility of C. albicans to drugs. We developed a new fluorescence method that allows the in vivo activity evaluation of compounds inhibiting of C. albicans transporters. We show that the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide diS-C3(3) is pumped out by both Cdr1 and Cdr2 transporters. The fluorescence labeling with diS-C3(3) enables a real-time observation of the activity of C. albicans Cdr1 and Cdr2 transporters. We demonstrate that enniatin A and beauvericin show different specificities toward these transporters. Enniatin A inhibits diS-C3(3) efflux by Cdr1 while beauvericin inhibits both Cdr1p and Cdr2p.

Keywords: ABC transporters; Candida albicans; beauvericin; diS-C3(3); enniatin A; inhibitors.

FIGURE 1

Fluorescence of diS-C₃(3) probe in strains with deleted transporters and wild-type strain. Probe was added at the beginning of the experiment and 2% glucose was added at the 60 min point. Fluorescence in the strains was measured every 4 min. The probe is exported by the ABC transporters Cdr2 and, after addition of glucose, Cdr1—but not by the MFS family transporter Mdr1 (N = 3–4).

FIGURE 2

Inhibition of ABC transporter Cdr1p by enniatin A. DiS-C₃(3) was added at the beginning of the experiment and fluorescence was measured every 4 min in strains with or without transporters; filled symbols indicate enniatin A addition (2 μg/ml) at 80 min, 2% glucose was added to the sample at 60 min (N = 3–4).

FIGURE 3

Inhibition of ABC transporters Cdr1p and Cdr2p by beauvericin. DiS-C₃(3) was added at the beginning of the experiment and fluorescence was measured every 4 min. Two percent glucose was added to the sample at 60 min and beauvericin at 80 min. (A) Influence of 1 μg/ml beauvericin on diS-C₃(3) staining in C. albicans WT and CDR1ΔCDR2Δ strains. (B) Dose-dependent inhibition of diS-C₃(3) staining by beauvericin on C. albicans CDR1Δ strain. Lower lambda max indicates more probe transported out of the cells. (C) Dose-dependent inhibition of diS-C₃(3) staining by beauvericin on C. albicans CDR2Δ strain.

FIGURE 4

Inhibition of diS-C₃(3) export by enniatin A and beauvericin. Staining was observed by confocal microscopy at 30 min (probe loaded into cells) and 150 min (full effect of the inhibitors).

FIGURE 5

Disk diffusion chemosensitization assays. Paper disks containing inhibitors (2 μg of enniatin A; 10 μg beauvericin; 1.25 μg fluconazole for WT, MDR1Δ, and CDR2Δ; 0.15625 μg fluconazole for the three remaining strains) were placed on the surface of agarose plates seeded with yeast and the plates were incubated at 28 °C for 48 h.