Isolation and Characterization of Fluoropyrimidine‐Resistant Mutants in <i>Two Candida</i> Species

Fasoli, M. O. F.; Kerridge, D.

doi:10.1111/j.1749-6632.1988.tb40411.x

Cited by 22 publications

(12 citation statements)

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“…The organisms used in these experiments were C. glabrata strain 4 (Whelan et al, 1984), C. albicans strain 72s (Whelan & Kerridge, 1984) and fluoropyrimidineresistant strains derived from them (Fasoli & Kerridge, 1988). Details of these strains are given in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

Fasoli¹,

Kerridge²

1990

Journal of General Microbiology

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The uptake of pyrimidines and their derivatives into Candida gZ&rata and Candiaiz albicans was measured using a novel technique in which the cells were rapidly separated from their suspending medium by centrifugation through a layer of an inert oil. The uptake of [' 4C]cytosine was linear for 30 s for all concentrations of pyrimidine tested. In The cytosine permease in C. gZabrata was specific for cytosine and 5-fluorocytosine. In C. albicans there was only one cytosine transport system [K, 2.4 & 0-3 p~; V50 f 4 pmol (pl cell water)-' s-l 1; this system also transported adenine, guanine and hypoxanthine. Differences in nucleoside transport were also observed for C. gZabrata and C. albicans, with the uridine permease in C. gZabrata transporting only uridine and 5-fluorouridine whereas cytidine and adenosine were also transported by the uridine permease in C. albicans. Studies on the effect of nucleoside analogues on uridine transport in C. gZabrata demonstrated the importance of the sugar moiety in determining the specificity of transport, with a hydroxyl residue on C-2 being apparently essential for transport.

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Section: Methodsmentioning

confidence: 99%

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

Fasoli¹,

Kerridge²

1990

Journal of General Microbiology

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“…After 3 and 5 h of incubation, 5-FU was barely visible in the intracellular pool, and the increase in the ratio of 5 gression of 5-FU metabolism, particularly between 1 and 3 h (Fig. 7); there is a corresponding increase in the levels of 5-FC and 5-FU in the supernatants; the ratio of these two signals remains roughly constant for up to 3 h of incubation, after which time it decreases as the extracellular concentration of 5-FC increases (Fig.…”

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confidence: 99%

“…"9F NMR spectra of 5 uridine permease-deficient strains of C. glabrata and C. albicans. The precise metabolic fate of 5-FUrd in C. glabrata and C. albicans is unclear (Fig.…”

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confidence: 99%

“…The choice of organism was dictated by the fact that C. glabrata is haploid (30) and therefore gives rise to a wider range of mutants resistant to 5-FC (and other fluorinated pyrimidines) than does the diploid C. albicans when subjected to UV mutagenesis and selection (5 The cells were then incubated with moderate agitation (110 rpm), and 20-ml samples were removed after 1, 3, or 5 h or as described below. For each time point of incubation, cells were harvested by centrifugation and the supernatant fluids were stored at -20°C.…”

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19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism

Fasoli

Kerridge

Morris

et al. 1990

Antimicrob Agents Chemother

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Flucytosine (5-FC)-resistant strains were isolated from the haploid opportunistic pathogen Candida glabrata by UV-induced mutation and fluoropyrimidine selection. These strains were characterized biochemically, and the metabolism of fluorinated pyrimidines was studied by '9F nuclear magnetic resonance spectroscopy. No evidence was obtained from these studies for degradative metabolism of the fluorinated derivatives. In the parental susceptible strain of C. glabrata, 5-fluorouracil but not 5-FC was detected within the cells. 5-Fluorouracil was also present in the culture supernatant after incubation of the cells with 5-FC. The distribution of fluorinated derivatives within the 5-FC-resistant strains was consistent with their genotype. Two strains of C. glabrata which had only a partial loss of cytosine deaminase and UMP pyrophosphorylase activity had high levels of resistance to 5-FC. Both C. glabrata and Candida albicans were susceptible to 5-fluorouridine. This compound but not the anticancer drug 5-fluoro-2-deoxyuridine was shown to be transported into susceptible cells by a specific uridine permease.Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for identifying drug metabolites (33) and investigating drug-receptor interactions in cell-free solutions (13). It can also be used noninvasively to study metabolism in vivo in microorganisms, isolated tissues, intact animals, or humans (18). The practical application of NMR spectroscopy in these living systems, however, is limited by sensitivity considerations.The "9F nucleus has the second-highest NMR sensitivity of the stable nuclei (ca. 85% of that of 'H at constant field). It is the naturally occurring isotope of fluorine (100% natural abundance) and is not present in living systems to any significant degree. The (6) and, more recently, in the liver (22, 31), plasma, and urine (8) of patients undergoing chemotherapy. In addition, it has been used to demonstrate tumor trapping of 5-FU in tumorbearing humans and rabbits (32). NMR spectroscopy has also been successful in demonstrating the existence of two distinct pathways of 5-FU degradation in E. coli (14).'9F NMR spectroscopy has also been employed in a preliminary study of the uptake and metabolism of the clinical antifungal drug 5-flucytosine (5-FC) in the medically important yeast Candida albicans (10). A similar but more quantitative approach was adopted to study 5-FC metabolism in three C. albicans strains and one Candida tropicalis * Corresponding author. strain (25). Results from these studies complemented each other and were consistent with the assigned genotypes of the strains used; in addition, they established the basic conditions under which "9F NMR spectroscopy may be used to obtain significant information on 5-FC metabolism in viable cells of Candida spp. Similar studies have been reported with Aspergillus spp. in which it has been shown that the main pathway of 5-FC metabolism is via the pyrimidine salvage pathway to 5-fluorouridine triphosphate (5-FUTP). In Aspergillus fumig...

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“…The possible mechanisms of resistance to 5-FC have been investigated, but most of this work was conducted prior to readily available molecular tools. Initial work employed UV mutagenesis in both C. albicans (4) and Saccharomyces 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.…”

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Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

Hope¹,

Tabernero

Denning³

et al. 2004

Antimicrob Agents Chemother

148

126

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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 ...

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Isolation and Characterization of Fluoropyrimidine‐Resistant Mutants in Two Candida Species

Cited by 22 publications

References 1 publication

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

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