Various cytosine/adenine permease homologues are involved in the toxicity of 5‐fluorocytosine in Saccharomyces cerevisiae

Abstract

5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil  ), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1 ), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues.

Mutational Analysis of Flucytosine Resistance in Candida glabrata

“…These data suggest that 5FC uptake is partially restricted in these Fcy2L mutants, conferring lowlevel resistance, but that cytosine uptake through the alternative Fcy2J or Fcy2C permease remains sufficient to confer growth. This is consistent with studies indicating multiple cytosine/5FC transporters in C. glabrata (4) and S. cerevisiae (19). 5FC-resistant clinical isolates demonstrate Fcy1, Fur1, and Fcy2J mutations.…”

“…This suggested a mutation in 5FC uptake. In S. cerevisiae, the purine-cytosine permease Fcy2 has been implicated in 5FC transport, although two other paralogs, Fcy21 and Fcy22, are present; the genes for all three are closely linked on a single chromosome (19,32). C. glabrata similarly encodes three Fcy2 homologs, although on separate chromosomes (L [Génole-vures accession no.…”

“…1). Studies of susceptible fungi, primarily the genetic model Saccharomyces cerevisiae, have shown that 5FC is taken up by one or more cytosine permeases (the most relevant is encoded by FCY2) (19,32) and modified to 5-fluorouracil (5FU) by cytosine deaminase (encoded by FCY1 [also known as FCA1 in C. albicans]) (3). Subsequent modifications to 5-fluoro-UMP by uracil phosphoribosyl transferase (UPRT; encoded by FUR1) (11,12) and to 5-fluoro-dUMP ultimately result in the disruption of protein and DNA synthesis.…”

“…Specifically, null mutants (including disruptants) of the genes encoding Fcy1, Fur1, and Fcy2 confer 5FC resistance (3,9,11,19). This is feasible because the salvage pathway employing these enzymes is nonessential, i.e., UMP can also be synthesized from exogenously acquired uridine or de novo from L-glutamine (Fig.…”

Molecular Mechanism of Flucytosine Resistance in Candida lusitaniae : Contribution of the FCY2 , FCY1 , and FUR1 Genes to 5-Fluorouracil and Fluconazole Cross-Resistance

“…Those affecting the FUR1 gene can be responsible for a 5FC-5FU cross-resistance pattern and can confer a very high level of resistance (MICs, Ն512 g/ml) to both drugs. Those affecting FCY1 or FCY2 confer a 5FC-FLC cross-resistance pattern and confer a lower level of resistance to 5FC (MICs, 64 to 128 g/ml) either because in fcy1 mutants 5FC can behave as an imperfect substrate of cytidine deaminase (2) or can be subjected to spontaneous nonenzymatic deamination (2) or because in fcy2 mutants 5FC can enter the cell through low-affinity permeases, as recently reported in S. cerevisiae (9).…”