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

Abstract

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...

Detection of FUR1 Gene in 5-Flucytosine Resistant Candida Isolates in Vaginal Candidiasis Patients

“…C. parapsilosis showed 3.4% resistance to itraconazole, while it showed 50% resistance in the present study. FUR 1 gene detection in 5FC resistant isolates correlated with findings of studies of Dodgson et al, [6] Hope et al, [20] and Pujol et al, [21].…”

“…However, large scale studies have shown primary resistance to C.albicans to be in the range of 3-6.5% [5]. Primary resistance to 5-flucytosine (5FC) occurs by decreased activity of either cytosine deaminase or UPRTase due to mutations in the genes, FCY1 or FUR1 [6,7].The study of Dodgson AR et al, showed single mutation at position 301 in FUR1 gene, responsible for 5FC resistance in C.albicans. In the present study, an attempt was made to detect the presence of mutant FUR1 gene in 5FC resistant Candida spp.…”

Molecular and Histological Association Between Candida albicans from Oral Soft Tissue and Carious Dentine of HIV-Positive Children

“…While antifungal resistance has been demonstrated to occur more frequently among clade II and clade SA isolates [20,21], conclusive evidence has yet to emerge of hyper-virulent strains through the use of accepted DNA fingerprinting techniques with high discriminatory power [22].…”

Characterization of the Same Mutations in FCA1 Gene Associated With 5-FC Resistance of Candida albicans

“…Mutations in the cytosine deaminase gene FCY1 (named FCA1 gene in C. albicans) or cytosine permease gene FCY2, which may result in the poor uptake of drug, are the most common causes of drug resistance (23,24). Primary resistance to 5-FC may also occur due to decreased activity of either cytosine deaminase or uracil phosphoribosyltransferase (UPRTase) as a result of mutations in the genes FCY1 or FUR1 (16,21,25,26). A recent research on gene FCA1 suggested that the presence of a Ser29Leu substitution in Fca1p in C. dubliniensis isolates is responsible for clade-specific resistance to 5-FC (27).…”

“…Amplification and sequencing of 5-FC resistanceassociated genes (FUR1, code for uracil phosphoribosyltransferase, and FCA1, code for cytosine deaminase) were also performed using a procedure described previously (16). The sequences of the two isolates were compared with the reference strain SC5314.…”