Fluconazole transport into Candida albicans secretory vesicles by the membrane proteins Cdr1p, Cdr2p, and Mdr1p

Abstract

A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [(3)H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [(3)H]fluconazole than did PGVs from empty-vector controls. The K(m)s (expressed in micromolar concentrations) and V(max)s (expressed in picomoles per milligram of protein per minute), respectively, for [(3)H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [(3)H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [(3)H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [(3)H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC(50)s; expressed as proportions of the [(3)H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

Fig. 1.

Effect of ACT1-regulated overexpression of CDR1, CDR2, or MDR1. (A) Intracellular [³H]fluconazole levels after incubation in 0.05 μM [³H]fluconazole for the times shown were highest in C. albicans DSY1050F cells transformed with pACT1 alone (empty vector), lower in C. albicans DSY1050F cells transformed with pACT1-CDR1 (CDR1), pACT1-CDR2 (CDR2), or pACT1-MDR1 (MDR1), and lowest in wild-type C. albicans SC5314. Data are means ± SD for 3 experiments. (B) Immunoreactive proteins of the sizes expected for Flag-tagged Cdr1p, Cdr2p, and Mdr1p, respectively, were demonstrated in Western blots of lysates of the pACT1-CDR1, pACT1-CDR2, and pACT-MDR1 transformants probed with anti-Flag antibodies but not in Western blots of lysates of pACT1-transformed controls (empty vector) (100 μg total protein per lane).

Fig. 2.

Effect of GAL1-regulated overexpression of CDR1, CDR2, or MDR1. (A) Fluconazole MICs were higher when C. albicans DSY1050F cells transformed with pGAL1-CDR1 (CDR1), pGAL1-CDR2 (CDR2), or pGAL1-MDR1 (MDR1) were incubated in galactose than when they were incubated in glucose, whereas the fluconazole MICs of pGAL-transformed controls (empty vector) were the same in galactose and glucose. (B) Intracellular [³H]fluconazole levels fell when 2% galactose was added to pGAL1-CDR1-, pGAL1-CDR2-, or pGAL1-MDR1-transformed C. albicans DSY1050F cells that had been incubated in YNB medium with 2% raffinose and 0.05 μM [³H]fluconazole until steady-state intracellular [³H]fluconazole levels were attained (16 h), but not when 2% galactose was added to pGAL1-transformed controls. Data are means ± SD for 3 experiments. (C) Immunoreactive proteins of the sizes expected for Flag-tagged Cdr1p, Cdr2p, and Mdr1p, respectively, were demonstrated in Western blots probed with anti-Flag monoclonal antibodies 2 h and 4 h after the pGAL1-CDR1, pGAL1-CDR2, and pGAL1-MDR1 transformants were exposed to galactose, but not in Western blots for controls exposed to raffinose or for pGAL1-transformed controls exposed to galactose or raffinose (100 μg total protein per lane).

Fig. 3.

Isolation of C. albicans post-Golgi vesicles (PGVs). (A) Transmission electron microscopy showed that membrane-bound PGVs accumulated in the cytoplasm when pACT1- and pS28N-transformed C. albicans DSY1050F cells were incubated for 7 h in 2% galactose, but not when they were incubated in 2% glucose. (B) In addition, there were many more intact PGVs in 100,000 × g pellets prepared from lysed spheroplasts of pACT1- and pS28N-transformed C. albicans DSY1050F cells incubated in galactose than in glucose-incubated controls. (C) Lastly, immunoreactive proteins of the sizes expected for Cdr1p, Cdr2p, and Mdr1p, respectively, were demonstrated by probing Western blots of the 100,000 × g pellets from C. albicans DSY1050F cells transformed with pS28N and either pACT1-CDR1 (CDR1), pACT1-CDR2 (CDR2), or pACT1-MDR1 (MDR1) with anti-Flag monoclonal antibodies, but not by probing Western blots of the 100,000 × g pellets prepared from pACT1-transformed controls (empty vector) (10 μg total protein per lane).

Fig. 4.

[³H]fluconazole accumulation by PGVs. (A) PGVs isolated from C. albicans DSY1050F cells transformed with pACT1-CDR1 (CDR1), pACT1-CDR2 (CDR2), or pACT1-MDR1 (MDR1) accumulated substantially more [³H]fluconazole after incubation in 0.05 μM [³H]fluconazole for the times shown than did PGVs from pACT1-transformed controls (empty vector). Data are means ± SD for 3 experiments. (B) Lineweaver-Burk plots of initial rates of [³H]fluconazole uptake by PGVs containing Cdr1p, Cdr2p, or Mdr1p.

Fig. 5.

Energy dependence of [³H]fluconazole transport. PGVs isolated from C. albicans DSY1050F transformed with pACT1-CDR1 (Cdr1p) or pACT1-CDR2 (Cdr2p) accumulated much more [³H]fluconazole after 10 s in gluconate buffer with ATP (GLU⁻) than in gluconate buffer without ATP (−ATP) or in gluconate buffer with ATP plus the nonhydrolyzable ATP analog AMP-PNP, the ATP inhibitor sodium orthovanadate (VAN), or the P-glycoprotein inhibitor verapamil (VER). In contrast, the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) had little effect on [³H]fluconazole transport by Cdr1p or Cdr2p. PGVs isolated from C. albicans DSY1050F cells transformed with pACT1-MDR1 (Mdr1p) accumulated much more [³H]fluconazole after 10 s in nitrate buffer (NO₃₋) than in gluconate buffer, and they accumulated much less [³H]fluconazole in nitrate buffer plus CCCP or in the presence of verapamil. In contrast, the absence of ATP or the presence of AMP-PNP or VAN had little effect on [³H]fluconazole transport by Mdr1p. Data are means ± SD for 3 experiments.

Fig. 6.

Inhibitors of [³H]fluconazole transport by Cdr1p, Cdr2p, and Mdr1p. [³H]fluconazole uptake by PGVs isolated from C. albicans DSY1050F cells transformed with pACT1-CDR1 (Cdr1p), pACT1-CDR2 (Cdr2p), or pACT1-MDR1 (Mdr1p) was quantified after incubation for 10 s in the presence of 50-fold molar excesses of the compounds listed. Data are means ± SD for 3 experiments. Am. Imidazole, 1-3-aminopropyl-imidazole; Cyt. Arabinose, cytosine β-d-arabinofuranoside.