Figure 6. Genetic Characterization of 5-FC and 5-FU in C. albicans .

(A) Pyrimidine salvage pathway, and transport and metabolism of 5-FC (see text for details).

(B) A hypothesis that fluorinated uracil blocks the formation of pseudouridine in rRNA. Note that pseudouridines are a prerequisite for ribosomal RNA processing [34].

(C and D) Functional characterization of potential permeases or transporters involved in the uptake of 5-FC (C) and 5-FU (D). A homozygous deletion strain for FCY2 (distinguished by the asterisk) and conditional shut-off (tetracycline repressible promoter, GRACE) strains [6] for genes indicated were tested in the absence (i.e., the non-repressing conditions, top panels) or the presence (i.e., the repressing conditions, bottom panels) of 100 μg/ml tetracycline against both compounds at the concentrations indicated. Note that 1) in (C) the FCY2 homozygous deletion strain was resistant to 5-FC, and 2) in (D) only the FCY21 GRACE strain exhibited slightly increased susceptibility to 5-FU under the non-repressing condition, suggesting that overexpression of FCY21 (from the tetracycline promoter, as observed in other cases, unpublished observations) may marginally facilitate the uptake of 5-FU.

(E) Suppression of 5-FC (top) and 5-FU (bottom) antifungal activity by genetic depletion of Fur1p. The FUR1 and HIS3 (the control) GRACE strains were grown in the presence (100 μg/ml, +TET) or absence (−TET) of tetracycline with 5-FC or 5-FU at the concentrations indicated. Individual growth was normalized with growth of the HIS3 strain without antifungal drug in the presence or absence of tetracycline. Note that when the expression of FUR1 was repressed, the strain was markedly resistant to both compounds, whereas it was modestly hypersensitive under the non-repressing conditions, likely due to overexpression of FUR1 from the tetracycline promoter. (The orf19 designations are as follows: FCY2 = orf19.1357, FCY21 = orf19.333, NNT1 = orf19.4118, DIP5 = orf19.2445, and FUR1 = orf19.2640.)