In Vitro Studies of 5-Fluorocytosine Resistance in Candida albicans and Torulopsis glabrata

104

Isolates of Cryptococcus neoformans from six patients were obtained before and after unsuccessful therapy with 5-fluorocytosine (5-FC). Post-therapy isolates exhibited massive and stable 5-FC resistance. The frequency of drug-resistant mutants in susceptible isolates of C. neoformans was <0.001% (70.4 ± 17.9 per 10 7 cryptococci), whereas mutant frequencies in resistant isolates approached 100%. Non-drug-induced, spontaneously appearing 5-FC resistant mutants were documented in four susceptible isolates of C. neoformans by use of the statistical method of fluctuation analysis. Mutation rates on these same four isolates ranged from 1.2 × 10 −7 to 4.8 × 10 −7 . Total intracellular uptake and incorporation of cytosine- 5 - 3 H (CyH 3 ) and 5-fluorocytosine- 2 - 14 C (5-FC 14 ) into a trichloroacetic acid-insoluble fraction were markedly reduced in six isolates with in vivo-acquired resistance when compared with susceptible pretreatment strains from the same patients. Five of these six isolates also had acquired massive resistance to 5-fluorouracil (5-FU), suggesting that a mutation in the uridine-5′-monophosphate pyrophosphorylase was responsible for drug resistance. The sixth isolate, which remained susceptible to 5-FU, appeared to have a defect in a cytosine-specific permease accounting for 5-FC resistance. A single isolate with in vitro-acquired 5-FC and 5-FU resistance had no reduction in uptake or incorporation of CyH 3 or 5-FC 14 . The mechanism of resistance in this isolate is discussed.

Section: Resultssupporting

confidence: 55%

5-Fluorocytosine Resistance in Cryptococcus neoformans

Block

Jennings

Bennett

1973

104

“…Insights into the mechanism of action of 5-FC in yeasts were provided in seminal papers by Jund and Lacroute (8), Kern et al (9,10), and Normark and Schonebeck (14). The present work draws upon these studies to investigate the molecular mechanisms of primary resistance to 5-FC in C. albicans.…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

Hope¹,

Tabernero

Denning³

et al. 2004

148

126

Primary resistance in Candida albicans to flucytosine (5-FC) was investigated in 25 strains by identifying and sequencing the genes FCA1, FUR1, FCY21, and FCY22, which code for cytosine deaminase, uracil phosphoribosyltransferase (UPRT), and two purine-cytosine permeases, respectively. These proteins are involved in pyrimidine salvage and 5-FC metabolism. An association between a polymorphic nucleotide and resistance to 5-FC was found within FUR1 where the substitution of cytidylate for thymidylate at nucleotide position 301 results in the replacement of arginine with cysteine at amino acid position 101 in UPRT. Isolates that are homozygous for this mutation display increased levels of resistance to 5-FC, whereas heterozygous isolates have reduced susceptibility. Three-dimensional protein modeling of UPRT suggests that the Arg101Cys mutation disturbs the quaternary structure of the enzyme, which is postulated to compromise optimal enzyme activity. A single resistant isolate, lacking the above polymorphism in FUR1, has a homozygous polymorphism in FCA1 that results in a glycine-to-aspartate substitution at position 28 in cytosine deaminase.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 activity but was subsequently found to possess antifungal activity and was used in 1968 to treat human cryptococcosis and candidiasis (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 treatment 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 pathway ( 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 transported into the cell by membrane permeases, 5-FC is converted via 5-FU to 5-fluoro-uridylate (synonymous with 5-fluoro-UMP ) 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-2Ј-deoxyuridylate, which inhibits the enzyme thymidylate synthetase and thus DNA synthesis by decreasing the available nucleotide pool. Mammalian cells lack the enzyme cytosine deaminase and consequently 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 observed in around 3% of isolates (1, 16). This phenomenon was recognized many years ago to be disproportionately represented in isolates belonging to serogroup B and more recently was found to ...

“…5-Fluorouridine monophosphate is then converted to 5-fluorouridine triphosphate, which, when incorporated into RNA in place of UTP, disrupts protein synthesis (7, 16). In addition, 5FU, when converted to 5-fluorodeoxyuridine monophosphate, disrupts DNA synthesis by inhibiting thymidylate synthetase (26).Although resistance to 5FC conceivably could develop by a number of mechanisms in C. albicans, it does so primarily by decreased activity of either cytosine deaminase or UPRTase (13,16,25). However, the mutations in the respective genes FCY1 and FUR1 responsible for resistance have not been identified.…”

mentioning

confidence: 99%

“…Although resistance to 5FC conceivably could develop by a number of mechanisms in C. albicans, it does so primarily by decreased activity of either cytosine deaminase or UPRTase (13,16,25). However, the mutations in the respective genes FCY1 and FUR1 responsible for resistance have not been identified.…”

mentioning

confidence: 99%

Clade-Specific Flucytosine Resistance Is Due to a Single Nucleotide Change in the FUR1 Gene of Candida albicans

Dodgson¹,

Dodgson

Pujol³

et al. 2004

121

Population studies have indicated that natural resistance to flucytosine (5FC) in Candida albicans is limited to one of the five major clades, clade I. In addition, while 73% of clade I isolates are less susceptible to 5FC (MIC > 0.5 g/ml), only 2% of non-clade I isolates are less susceptible. In order to determine the genetic basis for this clade-specific resistance, we sequenced two genes involved in the metabolism of 5FC that had previously been linked to resistance (cytosine deaminase and uracil phosphoribosyltransferase), in 48 isolates representative of all clades. Our results demonstrate that a single nucleotide change from cytosine to thymine at position 301 in the uracil phosphoribosyltransferase gene (FUR1) of C. albicans is responsible for 5FC resistance. The mutant allele was found only in group I isolates. The 5FC MICs for strains without copies of the mutant allele were almost exclusively <0.25 g/ml, those for strains with one copy of the mutant allele were >0.5 g/ml, and those for strains with two copies of the mutant allele were >16 g/ml. Thus, the two alleles were codominant. The presence of this allele is responsible for clade I-specific resistance to 5FC within the C. albicans population and thus by inference is likely to be the major underlying 5FC resistance mechanism in C. albicans. This represents the first description of the genetic mutation responsible for 5FC resistance.Analyses of the population structure of Candida albicans have revealed five major clades (I, II, III, SA, and E), each exhibiting a degree of geographical specificity (1,19,20,24). Recently, it was demonstrated that natural isolates that had been identified as resistant to flucytosine (5FC) were all members of clade I (18). Furthermore, it was demonstrated that 72% of clade I isolates exhibited reduced susceptibility to 5FC (5FC MIC Ն 0.5 g/ml) compared to 2% of non-clade I isolates (18). The fact that clades maintain their integrity side by side in the same geographical locale, combined with the observation that the majority of clade I isolates are less susceptible to 5FC, indicates that while recombination may occur within a clade, it may be a rare event between different clades (18,24), an interpretation consistent with earlier studies indicating that the population structure of C. albicans is primarily clonal (4,17).Although it has been demonstrated that natural resistance to 5FC is confined to isolates in clade I, the genetic basis of 5FC resistance has not been identified. 5FC enters a cell through the action of cytosine permease (15). Inside the cell, 5FC is converted to 5-fluorouracil (5FU) by cytosine deaminase, encoded by the gene FCY1 (7, 16). 5FU is then converted to 5-fluorouridine monophosphate by uracil phosphoribosyltransferase (UPRTase), encoded by the gene FUR1 (7, 16). 5-Fluorouridine monophosphate is then converted to 5-fluorouridine triphosphate, which, when incorporated into RNA in place of UTP, disrupts protein synthesis (7, 16). In addition, 5FU, when converted to 5-fluorodeoxyuridine monophosphat...