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. 2009 Nov 1;394(1):87-91.
doi: 10.1016/j.ab.2009.07.001. Epub 2009 Jul 3.

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Irena Ivnitski-Steele  1 , Ann R Holmes, Erwin Lamping, Brian C Monk, Richard D Cannon, Larry A Sklar
Affiliations

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Irena Ivnitski-Steele et al. Anal Biochem. .

Abstract

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.

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Figures

Fig. 1
Fig. 1
(A) R6G and (B) Nile Red content of S. cerevisiae control strain AD/pABC3 (vector only) and strains expressing C. albicans Cdr1p (AD/CDR1), Cdr2p (AD/CDR2), or Mdr1p (AD/MDR1) transporter genes. Yeast cells were incubated with 15 µM R6G (A) or 7 µM Nile Red (B) in DMSO (1%) for 20 min at 30°C. Enniatin (50 µM final) was added to strains preloaded with substrate and incubated for 20 min at 30°C. MCF: median channel fluorescence units. Each bar represents the median ± SD (n=3).
Fig. 2
Fig. 2
Dose-dependent Nile Red accumulation in control and pump-expressing strains. Pumpexpressing strains can efficiently efflux the substrate and maintain equilibrium at concentrations below 17 µM, while the control strain continuously accumulates Nile Red.
Fig. 3
Fig. 3
Dose-response analysis of Cdr1p, Cdr2p, and Mdr1p efflux activities to inhibition by enniatin (137 nM - 100 µM) using Nile Red as a substrate. Variable slope non-linear curve fit was applied for analysis.
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