Fluconazole assists berberine to kill fluconazole-resistant Candida albicans

Abstract

It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.

Fig 1

Time-kill curves of C. albicans strain 0304103 at different concentrations of FLC and BBR.

Fig 2

Subcellular localization of BBR. (A) The fluorescence of intracellular BBR was observed with excitation at 405 nm and emission at 520 nm. (B) The fluorescence of DAPI was observed with excitation at 364 nm and emission at 454 nm. (C) Fluorescence of intracellular BBR and DAPI. (D) Bright field. Scale bars = 5 μm.

Fig 3

(A) Cell cycle analysis by flow cytometry. C. albicans 0304103 cells were treated for 12 h or 24 h with 10 μg/ml of FLC (A1 and A3) or with 10 μg/ml of FLC plus 16 μg/ml of BBR (A2 and A4). Determination was performed in triplicate, and one representative experiment is shown. (B) Mean percentages of cells in G₁, S, and G₂. F, 10 μg/ml of FLC; FB, 10 μg/ml of FLC plus 16 μg/ml of BBR. The percentages in different phases were calculated by flow cytometry software. Statistical significance among groups was determined by analyses of variance. Comparison between the FLC monotreatment group and the FLC-BBR combination treatment group was performed by Student t test. The data are shown as means ± SDs from three independent experiments.

Fig 4

Chemical structures of selected DNA intercalators.

Fig 5

FLC increases the intracellular BBR concentration in C. albicans. C. albicans strain 0304103 was treated with different concentrations of FLC and BBR. (A) Fluorescence intensity was measured with an Infinite 200 Universal microplate reader at 405-nm excitation and 520-nm emission wavelengths over time. (B) Fluorescence intensity of the group treated with 16 μg/ml of FLC plus 4 μg/ml of BBR (B1) and the group treated with 4 μg/ml of BBR (B2) was measured at 0, 4, and 8 h (shown in the graph) by FACSCalibur cytometry. (C) Color of samples from different groups. Strains were photographed at 8 h.

Fig 6

Fluorescence intensity of samples treated with different concentrations of FLC and BBR for 8 h. Statistical significance among groups was determined by analyses of variance. Comparison between two groups was performed by Student t test. The data are shown as means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Fig 7

Ultrastructure of C. albicans strain 0304103. (A) Untreated control. (B) Cells treated with 16 μg/ml of BBR for 24 h. (C) Cells treated with 10 μg/ml of FLC for 24 h. (D) Cells treated with 10 μg/ml of FLC and 16 μg/ml of BBR for 24 h. White arrowhead, detachment of the cell membrane from the cell wall; white arrow, extensive solubilization in the cytoplasm.

Fig 8

(A) Efflux of intracellular BBR. Strains were treated with 16 μg/ml of FLC and 8 μg/ml of BBR for 8 h in RPMI 1640 medium, washed twice with PBS, and then resuspended with RPMI 1640 medium at 5 × 10⁷ cells/ml. With the addition of 2% glucose (Glu) or 16 μg/ml of FLC, BBR fluorescence measurement was performed with an Infinite 200 Universal microplate reader at 405-nm excitation and 520-nm emission wavelengths over time. (B and C) Intracellular BBR accumulation of different strains. Strains were treated with different concentrations of FLC and BBR, and fluorescence intensity was measured at 8 h. Statistical significance among groups was determined by analyses of variance. Comparison between two groups was performed by Student t test. The data are shown as means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Susceptibilities of different C. albicans strains to BBR. The strains included CAF2-1 (wild type), DSY448 (cdr1Δ/Δ), DSY653 (cdr2Δ/Δ), DSY465 (mdr1Δ/Δ), DAY294 (Tac1p normal-function strain), DAY296 (Tac1p gain-of-function mutant), DAY3706 (tac1Δ/Δ), and DAY3606 (tac1Δ/Δ::TAC1).