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Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans,10.1128/AAC.48.11.4377-4386.2004,Antimicrobial Agents and Chemotherapy,W

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans   (Citations: 21)
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Flucytosine (5-FC) was initially synthesized in 1957 as an anticancer drug. Unlike 5-fluorouracil (5-FU), a closely related fluorinated pyrimidine, 5-FC did not exhibit antineoplastic ac- tivity but was subsequently found to possess antifungal activity and was used in 1968 to treat human cryptococcosis and can- didiasis (25). Flucytosine administered in combination with amphotericin B remains the standard of care for cryptococcal meningitis, and the drug continues to have a role in the treat- ment of Candida infections which are life threatening or in circumstances where drug penetration may be problematic, such as infections of urine, eyes, and heart valves. Flucytosine is metabolized via the pyrimidine salvage path- way (Fig. 1), where it acts as a subversive substrate with the subsequent production of toxic nucleotides and disruption of DNA and protein synthesis (18, 26). After being actively trans- ported into the cell by membrane permeases, 5-FC is con- verted via 5-FU to 5-fluoro-uridylate (synonymous with 5-fluoro-UMP (5-FUMP)) under the action of the enzymes cytosine deaminase and uracil phosphoribosyltransferase (UPRT), respectively. 5-FUMP is in turn phosphorylated by two specific kinases to 5-fluoro-UTP, which is incorporated into RNA. 5-FUMP is also reduced to 5-fluoro-2-deoxyuridy- late, which inhibits the enzyme thymidylate synthetase and thus DNA synthesis by decreasing the available nucleotide pool. Mammalian cells lack the enzyme cytosine deaminase and con- sequently are not directly subject to the toxic effects of 5-FC. Primary resistance in Candida albicans, the focus of this paper, refers to inherent 5-FC resistance in the absence of prior drug exposure; in recent surveys, this resistance is ob- served in around 3% of isolates (1, 16). This phenomenon was recognized many years ago to be disproportionately repre- sented in isolates belonging to serogroup B and more recently was found to be confined to a specific clade defined by DNA fingerprinting (19). The possible mechanisms of resistance to 5-FC have been investigated, but most of this work was con- ducted prior to readily available molecular tools. Initial work employed UV mutagenesis in both C. albicans (4) and Saccha- romyces cerevisiae (8), and this work collectively demonstrated that disruption of any of the proteins involved in pyrimidine salvage or their regulation could lead to 5-FC resistance. We therefore considered it opportune to examine the molecular mechanisms of primary resistance in C. albicans by identifying and sequencing the genes coding for the proteins involved in pyrimidine salvage and analyzing the effect of one of these polymorphisms using protein modeling. The findings in this paper are consistent with a recently published study in which the majority of cases of 5-FC resis- tance in C. albicans were associated with isolates that were homozygous for a single amino acid substitution, Arg101Cys, in UPRT (2). The present paper explores the relationship of this mutation to an analogous mutation in the UPRT of S. cerevisiae and provides a structural basis by which the mutation induces resistance. It is clear, nevertheless, that this substitu- tion cannot fully account for the level of 5-FC resistance seen in most isolates. This study also implicates an amino acid substitution within cytosine deaminase and further explores the association of substitutions in the purine-cytosine per- meases with 5-FC resistance. (This work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chi- cago, Ill., 14 to 17 September 2003.)
Journal: Antimicrobial Agents and Chemotherapy - ANTIMICROB AGENTS CHEMOTHER , vol. 48, no. 11, pp. 4377-4386, 2004
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