Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Abstract

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Keywords: ABC transport; Afr1; Afr2; Mdr1; azoles; efflux pump.

FIG 1

Deletion of efflux pump genes affects drug resistance levels. The MICs of different compounds in each indicated strain were determined (red circles, H99 and its derivatives; black squares, R265 and its derivatives). The experiments were repeated three times, and the bars represent the average MICs. The details for each strain's MICs are listed in Table 1. The pictures on the right side of panel A are the fluconazole Etest results from a representative experiment after 3 days of incubation. FLC, fluconazole; VRC, voriconazole; ITC, itraconazole; CHX, cycloheximide; R6G, rhodamine 6G; TSA, trichostatin A.

FIG 2

Deletion of efflux pump genes affects drug accumulation. (A) Wild-type and deletion strains were incubated with 10 μM R6G (A and B) or 7 μM Nile Red (C and D) in YEPD medium for 30 min at 30°C, and the cells were analyzed by flow cytometry. (A and C) H99; (B and D) R265. Values represent the means ± standard deviations from three independent experiments. *, P < 0.0442; ***, P < 0.0006; ****, P < 0.0001 (Holm-Sidak's multiple-comparison test).

FIG 3

Deletion of efflux pump genes influences the formation of disomic chromosomes. Quantitative PCR from single colonies was performed using FLC-resistant clones derived from the indicated strains. Probes specific for different chromosomes were chosen to assess the changes in genomic copy number. The PCR results with the Chr1- and Chr5-specific probes were compared to those with the Chr3-specific probe, which served as unduplicated internal control in the indicated strains. (A) Copy number of the gene on Chr1. (B) Copy number of the gene on Chr5. Values represent the means ± standard deviations for four independently isolated FLC-resistant clones of each mutant.

FIG 4

Gene expression levels of AFR1, AFR2, and MDR1. Cells of the indicated strains from the exponential growth phase were either untreated (NT) or treated (FLC) with 32 μg/ml FLC for 2 h. RNA was isolated, and the expression levels of each gene were determined by qRT-PCR. The expression levels of each gene under each condition were normalized to that of the actin gene and compared to the expression levels of the wild type without FLC treatment. (A, B, and C) Expression levels of AFR1, AFR2, and MDR1, respectively, in H99. (D, E, and F) Expression levels of AFR1, AFR2, and MDR1, respectively, in R265. Values represent the means ± standard deviations for three biological replicates. **, P < 0.0091; ***, P < 0.0007; ****, P < 0.0001 (uncorrected Fisher's least significant difference [LSD]).

FIG 5

Expression of efflux pump genes and ERG11 is independently regulated by different transcription factors. Exponential-growth-phase cells of transcription factor mutant strains were either untreated (NT) or treated (FLC) with 32 μg/ml FLC for 2 h. RNA was isolated, and the expression levels of each gene were determined by qRT-PCR. The expression levels of each gene were normalized to that of the actin gene and compared to the expression levels of the wild type without FLC treatment. (A) Expression levels of CnAFR1; (B) expression levels of CnAFR2; (C) expression levels of CnMDR1; (D) expression levels of CnERG11. Values represent the means ± standard deviations for three biological replicates. *, P < 0.0476; **, P < 0.0098; ***, P < 0.0006; ****, P < 0.0001 (uncorrected Fisher's LSD). The details for the expression levels for each gene in the FLC-resistant mutants and mutants with increased frequencies of FLC heteroresistance are shown in Fig. S1 in the supplemental material.