| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany
Correspondence
Friedhelm Meinhardt
meinhar{at}uni-muenster.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
fcy1 mutants, lacking cytosine deaminase, became entirely resistant to 5FC, concomitantly losing 5FC+FLC additivity. Disruption of the orotate phosphoribosyltransferase gene (URA5) in the wild-type led to low-level 5FC tolerance, while an alternative orotate phosphoribosyltransferase, encoded by URA10, contributed to 5FC toxicity only in the ura5 background. Remarkably, combination of ura5 and fur1 resulted in complete 5FC resistance. Thus, yeast orotate phosphoribosyltransferases are involved in 5FC metabolism. Similarly, disruption of the ergosterol 5,6-desaturase-encoding gene ERG3 resulted only in partial resistance to FLC, and concomitantly a synergistic effect with 5FC became evident. Full resistance to FLC occurred in erg3 erg11 double mutants and, simultaneously, synergism or even an additive effect with FLC and 5FC was no longer discernible. Since the majority of spontaneously occurring resistant yeast clones displayed residual sensitivity to either 5FC or FLC and those strains responded to combined drug treatment in a predictable manner, careful resistance profiling based on the findings reported here may help to address yeast infections by combined application of antimycotic compounds.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). Fluconazole (FLC) on the other hand, exhibits a completely different mode of action, targeting Erg11, essential for the production of ergosterol in fungi (Kalb et al., 1987; Vanden Bossche, 1985). These two agents are routinely administered in combination to combat a broad spectrum of fungal infections (Mukherjee et al., 2005; Johnson et al., 2004). The desired effect of such combination therapy is an additive or synergistic increase in antifungal activity when compared to singly applied compounds. In a previous study, Saccharomyces cerevisiae was used as a model organism to identify putative permeases which play a role in 5FC toxicity. In S. cerevisiae, 5FC uptake is brought about mainly by the cytosine permease Fcy2; however, in its absence, several other permeases ensure residual 5FC influx (Paluszynski et al., 2006). However, the impact of disruptions in the 5FC metabolic pathway on synergy when combined with FLC has yet to be studied. Interestingly, in Cryptococcus neoformans, synergism was observed in cases in which primary resistance to one of the agents occurred (Schwarz et al., 2003, 2006, 2007), but the reasons remained unknown. Depending on the agent and yeast species, however, adverse (antagonistic) effects of a combination therapy are also documented (Te Dorsthorst et al., 2002). The outcome of a combined antimycotic treatment apparently varies within genera, species and even isolates, and is thus hardly predictable. Phylogenetic analyses of pathogenic yeast species indicated that S. cerevisiae is closely related to the major opportunistic fungal pathogen Candida albicans (Barns et al., 1991; Hendricks et al., 1989; Lupetti et al., 2002). Thus, despite the above constraints, S. cerevisiae may serve as a valuable model to study antifungal drug action (Agarwal et al., 2003).
5FC is a prodrug, which to exert its action requires uptake and metabolism to either 5-fluorouridine triphosphate (5FUTP, formed from 5FUDP; see Fig. 1) or 5-fluorodeoxyuridine monophosphate (5FdUMP), the former directly disturbing transcription and the latter – via inhibition of thymidylate synthetase and subsequent dTTP depletion – aborting DNA synthesis (Polak & Scholer, 1975; Hartmann & Heidelberger, 1961; Whelan, 1987; Wadler et al., 1998). Essential steps in intracellular 5FC metabolism are the conversion to 5-fluorouracil (5FU) by the cytosine deaminase Fcy1 and subsequent processing to 5-fluorouridine monophosphate by the uracil phosphoribosyltransferase Fur1 (Chevallier et al., 1975; Vanden Bossche et al., 1994, 1987; Kern et al., 1990; Kurtz et al., 1999).
|
), explaining the known rapid establishment of spontaneous resistance (Alexander & Perfect, 1997; Vanden Bossche et al., 1987; Paluszynski et al., 2006).
FLC and several other azole antimycotics interfere with the biosynthesis of ergosterol, a fungal-specific sterol that is important for membrane integrity (Smith et al., 1996, Bammert & Fostel, 2000). The specific target of antimycotic azoles is the lanosterol 14
-demethylase, encoded by the ERG11 gene (Kalb et al., 1987; Vanden Bossche, 1985). By binding the iron atom of the haem moiety, activation of oxygen, necessary for demethylation of lanosterol, is prevented (Joseph-Horne & Hollomon, 1997), eventually resulting in the accumulation of toxic 14-methylergosta-8,24(28)-dien-3β,6-diol (14-methyl-3,6-diol), which impairs membrane function (Kelly et al., 1995).
Mechanisms of azole resistance in S. cerevisiae and C. albicans include overproduction or alteration of Erg11p, or modification of downstream enzymes (Bard et al., 1993; Vanden Bossche et al., 1992; Hitchcock, 1991). The loss of function of the sterol 5,6-desaturase encoded by ERG3 results in accumulation of episterol, which permits fungal growth in the presence of azole drugs (Arthington et al., 1991; Watson et al., 1988; Kelly et al., 1995). However, disruption of the aforementioned ERG11 gene was shown to be lethal in S. cerevisiae, as accumulation of 14-methyl-3,6-diols facilitates growth only under anaerobic conditions and in ergosterol-supplemented media (Watson et al., 1989; Bard et al., 1993). This growth arrest can be circumvented by the inactivation of ERG3, which results in the accumulation of methylfecosterol instead of 14-methyl-3,6-diol. In contrast, C. albicans ERG11 null mutants are capable of survival in the absence of a suppressor mutation in ERG3, albeit with a severe growth defect (Sanglard et al., 2003). Hence, C. albicans either produces different levels of the diol and/or is less sensitive to its lethal effects as compared to S. cerevisiae (Watson et al., 1989; Bard et al., 1993).
To determine the prerequisites underlying the synergistic or additive increase in antifungal action of 5FC/FLC combinations, we set up a S. cerevisiae model system consisting of an isogenic strain collection with combinations of defined deletions in genes important for 5FC and/or FLC toxicity. Sensitivity profiling revealed residual drug responses and additive action of the two drugs in the majority of strains despite the establishment of primary resistance. The genetic basis of such residual activity was investigated and identified as being essential for increased drug action in combined applications of 5FC and FLC.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
. Cultivation was performed in complete YPD (1 %, w/v, Bacto-yeast extract, 2 %, w/v, Bacto-peptone, 2.2 %, w/v, glucose) or YNB (Yeast Nitrogen Base, Difco) media supplemented with (L-leucine 10 µg ml–1, L-methionine 10 µg ml–1, uracil 200 µg ml–1, uridine 100 µg ml–1) when required. Media were prepared according to Kaiser et al. (1994). Strains were cultivated at 30 °C in YPD liquid or on agar medium. Escherichia coli strains were grown in LB (1 %, w/v, peptone, 0.5 %, w/v, yeast extract, 0.5 %, w/v, NaCl, pH 7.3) at 37 °C (Sambrook et al., 1989) with a working concentration of ampicillin of 100 µg ml–1.
|
. Restriction and ligation of DNA was carried out according to the suppliers' recommendations (NEB and MBI-Fermentas). For Southern analyses (Southern, 1975) DNA fragments were labelled by applying the DIG-DNA-labelling and detection kit from Roche Biochemicals.
Transformation and gene disruptions.
E. coli JM109 was transformed by the CaCl2 method as described by Sambrook et al. (1989). Transformants of S. cerevisiae were obtained according to Gietz & Schiestl (1995), and selected on YNB agar.
The ERG3 gene was disrupted using the KlLEU2 marker gene cloned in pUG73. ERG3 was amplified and blunt-end inserted into the HincII site of the pSK plasmid vector (see Table 2 for primers). Subsequently, the KlLEU2 gene from pUG73 was excised (HincII and PvuII) and inserted into the internal ERG3 HincII site. The disruption cassette erg3 : : LEU2 was introduced into S. cerevisiae BY4741, S. cerevisiae fcy1 and S. cerevisiae fcy2 strains (Table 1
). Mutants obtained from EUROSCARF (Frankfurt, Germany), fcy1 and fcy2, were verified using primer pairs Fcy1F/R and Fcy2F/R, respectively. Homologous recombination with the ERG3 disruption cassette was checked by PCR, in a Mini-Cycler (MJ Research Biozym) and Southern analysis.
|
; Gueldener et al., 2002). For URA5 disruption, primers (URA5koF and URA5koR) flanking the SpHIS5 marker gene from pUG27 and an additional 45 bp homologous to URA5 were used. URA10 was disrupted using the KlLEU2 marker gene from pUG73, with primers URA10koF and URA10koR. FUR1 was disrupted using Fur1koF and Fur1koR primers flanking the KlURA3 marker gene. The URA5 : : SpHIS5 knockout cassette was transformed into S. cerevisiae fur1 and ura10, resulting in ura5 fur1 and ura5 ura10 double mutant strains. Disruption of URA5 was PCR verified, employing primer pairs URA5outR/HIS5up and URA5outF/HIS5down, respectively. For verification of URA10 and FUR1 knockouts, primer pairs LEU2down/URA10outF, LEU2up/URA10outR and URA3down/Fur1outF, URA3up/Fur1outR were applied.
In vitro drug susceptibility tests.
Stock solutions of 5FC and FLC obtained from MP Bio-medicals, Frankfurt, Germany, were established with a final concentration of 5 mg ml–1, and after sterile-filtration (cellulose acetate membrane, pore size 0.2 µm; Millipore), stored at –20 °C. Prior to use drugs were added to YPD agar at approximately 50 °C to establish appropriate concentrations. For testing resistance rapidly, cultures were diluted 10–1 to 104 and 8 µl of each dilution was spotted onto the agar plates. For proper quantification, 200 µl volumes of liquid YPD medium containing 5FC, FLC, and both in combination, were inoculated with 1x106 yeast cells and incubated at 30 °C in U-profile 96-well microtitre plates (Carl Roth) for 24 h; growth was monitored spectroscopically at 600 nm as previously described (Paluszynski et al., 2006).
Where applicable, the minimal inhibitory concentration (MIC) for 5FC and FLC was read as the lowest drug concentration that gave 50 % or more growth inhibition, which is defined as MIC-2 as described by the National Committee for Clinical Laboratory Standards (NCCLS, 1998). The fractional inhibitory concentration (FIC) was calculated to quantify drug interaction (Elfopouios & Moellering, 1991), being defined as synergistic if the FIC was 
0.5, additive if FIC was >0.5 and 1, indifferent if 1 <FIC 4, and antagonistic if FIC was >4.
The FIC index was determined as follows: FIC=[(MIC of drug A, in combination)/(MIC of drug A, tested alone)]+[(MIC of drug B, in combination)/(MIC of drug B, tested alone)].
Measurement of growth of erg3 and erg3 erg11 mutants.
To measure the growth of erg3 and erg3 erg11 mutant strains, in comparison to the wild-type, growth curves were established by inoculating 1 % of overnight pre-cultures in 50 ml YPD medium. Cultivation was performed in the presence and absence of FLC at 30 °C, with the final concentration of FLC being 1 mg ml–1. Growth was monitored spectroscopically at 600 nm until strains reached the stationary phase.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
); FLC resistance was achieved by disruption of ERG3, encoding the C-5 sterol desaturase.
Resistance levels of respective mutants of the BY4741 background were initially monitored using a drop dilution plate assay employing various concentrations of 5FC and FLC. As to be expected, fcy1, fcy2 and fur1 mutations clearly conferred 5FC resistance (Fig. 2a), whereas FLC resistance was solely observed in the erg3 strain (Fig. 2b). There was no cross-resistance to FLC in fcy1, fcy2 or fur1 strains and vice versa; as for the wild-type 5FC sensitivity was seen in the erg3 mutant.
|
). Neither in wild-type nor in any of the mutant strains were antagonistic effects of combined 5FC/FLC application observed. Rather, clear additive interactions of both drugs were detected in the wild-type (FIC=0.74) and the fcy2 mutant (FIC=0.576) or synergism in the case of the erg3 mutant (FIC=0.20), despite the fact that the latter two exhibit resistance to 5FC or FLC (Figs 3 and 4a). Such an increase in efficiency for 5FC+FLC was also seen in the fur1 mutant (FIC=0.63) (Fig. 3d). Significantly, there is a remarkable difference in the residual responses to the singly applied antimycotics that correlates with additive or synergistic interactions with the second drug. Despite the response being clearly distinct in strains deficient in either FCY2 or FUR1, both show clear growth inhibition by 5FC, dose-dependent in fcy2 and dose-independent in the fur1 mutant (Fig. 3). Similarly, there is an apparent FLC-dependent growth retardation in the erg3 strain, which is, however, not aggravated with increasing FLC concentrations (Fig. 4a). By comparing growth of wild-type and erg3 cells, a general growth-affecting defect in erg3 became obvious; however with FLC, growth was additionally retarded (Fig. 4b). Thus, FLC inhibits growth even in cells carrying the FLC resistance mutation erg3.
|
|
). The Erg3 FLC-toxicity-promoting reaction is the generation of a toxic diol from 14-methylfecosterol, the latter being formed from lanosterol by inhibition of the 14- demethylase (Erg11) by FLC (Bard et al., 1993; Watson et al., 1989). Thus, erg3 mutants tolerate FLC, as no toxic diols are synthesized, but concomitantly exhibit an altered membrane sterol composition probably accounting for the observed slow growth of these mutants (see also Fig. 4).
Although the main target of FLC, Erg11, is normally essential, it is dispensable when Erg3 is additionally knocked out, as this abrogates formation of toxic diols (Bard et al., 1993; Watson et al., 1989). erg3 erg11 cells displayed full FLC resistance, but again, the strong FLC-independent growth defect became obvious (Fig. 4a, c). Interestingly, growth of the double mutant (erg3 erg11), irrespective of the presence or absence of FLC, is comparable to that of the erg3 strain with FLC. In each of the latter, the ergosterol biosynthesis pathway yields 14-methylfecosterol, evidently causing a more pronounced growth impairment than episterol but less than 14-methyl-3,6-diol formed in wild-type cells when exposed to FLC (Fig. 4d). Importantly, synergistic actions of FLC and 5FC, as seen in erg3 cells, are cancelled by the additional erg11 mutation, indicating the requirement of at least residual FLC-mediated growth retardation for synergistic or additive drug action.
As for the erg3 erg11 mutant, the additive response was abolished when FCY1 was lacking, with both displaying an FIC of 1 and, at least consistent with residual sensitivity being crucial for additive action, fcy1 mutants displayed no response to 5FC regardless of the concentration applied (Fig. 3b). Thus, synergistic or additive antifungal action of 5FC/FLC treatment is seen in erg3, fcy2 and fur1 strains, whereas such an effect is absent in erg3 erg11 double and fcy1 single mutants, which do not respond to either FLC or 5FC, respectively.
5FC response in fur1 mutants
We have recently shown that toxicity of 5FC in fcy2 mutants is due to the presence of several other permeases capable of low-level 5FC transport in S. cerevisiae (Paluszynski et al., 2006). Growth inhibition of fur1 mutants by 5FC must, however, occur in a different manner, since the Fur1-catalysed conversion of 5FU to 5FUMP is crucial for downstream effects of 5FC (Fig. 1). Unlike mammals, S. cerevisiae lacks alternative enzymes capable of 5FU metabolism, such as thymidine or uridine phosphorylase (Jund & Lacroute, 1970). According to the Candida genome database (http://www.candidagenome.org/) and Cryptococcus neoformans genome project (http://www.tigr.org/tdb/e2k1/cna1/), there is also no evidence for the presence of thymidine or uridine phosphorylases in these genera. In mammals, 5FU can also be metabolized by orotate phosphoribosyltransferase, an enzyme involved in de novo pyrimidine biosynthesis (Peters et al., 1984); the engagement of the respective yeast enzymes in 5FC antimycotic activity has, however, yet to be investigated. S. cerevisiae possesses two homologous orotate phosphoribosyltransferases, Ura5 and Ura10 (Fig. 5a), which are only distantly related to the functional analogues of mammals (de Montigny et al., 1990). Hence, the involvement of such yeast orotate phosphoribosyltransferases in 5FU metabolism was checked by disrupting URA5 and URA10 singly and in combination. Effects on 5FC tolerance were subsequently recorded by the microtitre plate assay (Fig. 5b). Indeed, the ura5 single mutant displayed moderate resistance at low 5FC concentrations (Fig. 5b). However, ura10 alone did not affect 5FC tolerance significantly, but slightly contributed to resistance in the ura5 background (not shown). Most remarkably, however, full dose-independent 5FC resistance was established when URA5 was disrupted in the fur1 strain (Fig. 5b). Thus, both Ura5 and Ura10 are capable of 5FU metabolism and probably mediate residual 5FC toxicity in the absence of Fur1. As homologues of S. cerevisiae Ura5/10 have been identified in a variety of ascomycetous yeast species, including Candida albicans and C. glabrata, and also in the basidiomycetous yeast Cryptococcus neoformans (Fig. 5a), it appears probable that a requirement for uracil phosphoribosyltransferase in 5FC prodrug activation and antifungal activity can generally be bypassed to some extent by orotate phosphoribosyltransferases. Among three loci known to be involved in 5FC uptake and activation (FCY1, FCY2 and FUR1) in S. cerevisiae, only one proved to be essential (FCY1). It has previously been shown that S. cerevisiae fcy1 mutants entirely lack cytosine deaminase activity, and fcy1 ura3 or ura2 double mutants, which are additionally defective in de novo pyrimidine synthesis, are unable to grow with exogenously supplied cytosine (Jund & Lacroute, 1970; Erbs et al., 1997), clearly excluding an alternative enzyme for bypassing the Fcy1 deamination of cytosine or 5FC. In contrast, Fcy2 and Fur1 reactions can be catalysed by structurally or functionally related proteins, thereby explaining successful application of 5FC in instances where (partial) resistance was established by targeted gene disruption in the model system, or by spontaneous mutation in resistant clinical isolates.
|
; de Kruijff & Demel, 1974) might restore 5FC penetration, thus explaining synergistic antifungal activity in a cytosine-permease-defective strain (Schwarz et al., 2007). However, synergism was also observed in 5FC-resistant isolates when this agent was combined with FLC, which does not directly affect membrane integrity (Allendoerfer et al., 1991). As we detected additive action of 5FC/FLC not only in fcy2 mutants of S. cerevisiae but also in a fur1 strain, which is fully capable of 5FC import, we suggest that aided 5FC penetration by membrane-damaging antimycotics may increase synergism in 5FC-resistant strains, but is probably not generally required.
Effects of 5FC and FLC on spontaneously occurring resistant clones
To elucidate whether residual antimycotic response in spontaneous mutants is indeed detectable and functionally linked with susceptibility to combined 5FC/FLC application, naturally occurring drug-resistant mutants in S. cerevisiae were screened. Among 73 clones obtained in a screening for 5FC tolerance, 39 turned out to be stable. Almost all of the latter displayed additive effects when treated jointly with both drugs; only three of them eventually turned out to be fully resistant, and interestingly, for these isolates, the 5FC/FLC combination had the same effect as the singly applied 5FC (Table 3). All strains obtained in a screening for FLC tolerance (n=36) still displayed residual drug sensitivity. Remarkably, combined application of both antimycotic compounds increased their biological activities in either case, supporting the notion that residual drug response is required and sufficient for additive antifungal activity of the 5FC/FLC combination (Table 3). It is noteworthy that occurrence of full resistance to either drug was an exception rather than the rule in our experiments. In fact we have experienced it only for the three strains being fully resistant to 5FC. In all other instances susceptibility to combined drug treatment was prevalent.
|
) or phosphoribosyltransferases (Fig. 5b), respectively. The finding that primary resistance does not generally abolish the effectiveness of 5FC, in particular when combined with FLC, may support the use of this agent in clinical applications despite the known rapid establishment of spontaneous resistance. Clearly, however, when 5FC resistance is due to loss of function of the cytosine deaminase, 5FC application is not appropriate.
Testing combined drug efficiency in defined mutants with primary resistance to 5FC and FLC
Double mutants carrying mutations conferring resistance to both agents were generated to check whether partial resistance to either 5FC or FLC still allows efficient combined drug action (Fig. 6). Indeed, combination of the erg3 mutation with either fcy2 or fur1 resulted in strains displaying robust resistance to both 5FC and FLC. However, as for the respective single mutants, residual response to both 5FC and FLC was observed in the erg3 fcy2 (FIC=0.57) and the erg3 fur1 strain, where a distinct FIC could not be determined (Fig. 6a, b). Combined application of both drugs led to increased antifungal activity; thus even a primary resistance against two antifungal agents does not a priori exclude additive effects in combinational treatments. Combining full 5FC (fcy1) with partial FLC (erg3) resistance, however, completely abolished additivity, supporting the conclusion that at least a faint residual response to both of the drugs is required (Fig. 6a).
|
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. Pla
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Alexander, B. D. & Perfect, J. R. (1997). Antifungal resistant trends towards the year 2000: Implications of therapy and new approaches. Drugs 54, 657–678.[Medline]
Allendoerfer, R., Marquis, A. J., Rinaldi, M. J. & Graybill, J. R. (1991). Combined therapy with fluconazole and flucytosine in murine cryptococcal meningitis. Antimicrob Agents Chemother 35, 726–729.
Arthington, B. A., Bennett, L. G., Skatrud, P. L., Guynn, C. J., Barbuch, R. J., Ulbright, C. E. & Bard, M. (1991). Cloning, disruption, and sequence of the gene encoding yeast C-5 sterol desaturase. Gene 102, 39–44.[CrossRef][Medline]
Bammert, G. F. & Fostel, J. M. (2000). Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrob Agents Chemother 44, 1255–1265.
Bard, M., Lees, N. D., Turi, T., Craft, D., Cofrin, L., Barbuch, R., Koegel, C. & Loper, J. C. (1993). Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids 28, 963–967.[Medline]
Barns, S. M., Lane, D. J., Sogin, M. L., Bibeau, C. & Weisburg, W. G. (1991). Evolutionary relationships among pathogenic Candida species and relatives. J Bacteriol 173, 2250–2255.
Chevallier, M. R., Jund, R. & Lacroute, F. (1975). Characterization of cytosine permeation in Saccharomyces cerevisiae. J Bacteriol 122, 629–641.
de Kruijff, B. & Demel, R. A. (1974). Polyene antibiotic–sterol interactions in membranes of acholeplasma-laidlawii cells and lecithin liposomes. 3. Molecular structure of polyene antibiotic–cholesterol complexes. Biochim Biophys Acta 339, 57–70.[Medline]
de Kruijff, B., Gerritsen, W. J., Oerleman, A., Demel, R. A. & Van Deenen, L. L. M. (1974). Polyene antibiotic–sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. 1. Specificity of membrane-permeability changes induced by polyene antibiotics. Biochim Biophys Acta 339, 30–43.[Medline]
de Montigny, J., Kern, L., Hubert, J. C. & Lacroute, F. (1990). Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae. Curr Genet 17, 105–111.[CrossRef][Medline]
Elfopouios, G. M. & Moellering, R. C., Jr (1991). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 3rd edn, pp. 432–492. Edited by U. Lorian. Baltimore, MD: Williams & Wilkins.
Erbs, P., Exinger, F. & Jund, R. (1997). Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans. Curr Genet 31, 1–6.[CrossRef][Medline]
Fasoli, M. O., Kerridge, D., Morris, P. G. & Torosantucci, A. (1990). 19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism. Antimicrob Agents Chemother 34, 1996–2006.
Ghannoum, M. A. & Rice, L. B. (1999). Antifungal agents mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12, 501–517.
Gietz, R. D. & Schiestl, R. H. (1995). Transforming yeast with DNA. Methods Mol Cell Biol 5, 255–269.
Gueldener, U., Heinisch, J., Koehler, G. J., Voss, D. & Hegemann, J. H. (2002). A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30, e23
Hartmann, K. U. & Heidelberger, C. (1961). Studies on fluorinated pyrimidines. XIII. Inhibition of thymidylate synthetase. J Biol Chem 236, 3006–3013.
Hendriks, L., Goris, A., Neefs, J., van de Peer, Y., Hiennebert, G. & de Wachter, R. (1989). The nucleotide sequence of the small ribosomal subunit RNA of the yeast Candida albicans and the evolutionary position of the fungi among the eukaryotes. Syst Appl Microbiol 12, 223–229.
Hitchcock, C. A. (1991). Cytochrome P-450-dependent 14-sterol demethylase of Candida albicans and its interaction with azole antifungals. Biochem Soc Trans 19, 782–787.[Medline]
Johnson, D. M., MacDougall, C., Ostrosky-Zeichner, L., Perfect, J. R. & Rex, J. R. (2004). Combination antifungal therapy. Antimicrob Agents Chemother 48, 693–715.
Joseph-Horne, T. & Hollomon, D. W. (1997). Molecular mechanisms of azole resistance in fungi. FEMS Microbiol Lett 149, 141–149.[CrossRef][Medline]
Jund, R. & Lacroute, F. (1970). Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Saccharomyces cerevisiae. J Bacteriol 102, 607–615.
Kaiser, C., Michaelis, S. & Mitchell, A. (1994). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R. & Loper, J. C. (1987). Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA 6, 529–537.[Medline]
Kelly, S. L., Lamb, D. C., Corran, A. J., Baldwin, B. C. & Kelly, D. E. (1995). Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3-beta,6-alpha-diol. Biochem Biophys Res Commun 207, 910–915.[CrossRef][Medline]
Kern, L., de Montigny, J., Jund, J. & Lacroute, F. (1990). The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene 88, 149–157.[CrossRef][Medline]
Kurtz, J. E., Exinger, F., Erbs, P. & Jund, R. (1999). New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism. Curr Genet 36, 130–136.[CrossRef][Medline]
Lupetti, A., Danesi, R., Campa, M., Del Tacca, M. & Kelly, S. (2002). Molecular basis of resistance to azole antifungals. Trends Mol Med 8, 76–81.[CrossRef][Medline]
Mukherjee, P. K., Sheehan, D. J., Hitchcock, C. A. & Ghannoum, M. A. (2005). Combination treatment of invasive fungal infections. Clin Microbiol Rev 18, 163–194.
NCCLS (1998). Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi: proposed standard M38-P. Wayne, PA: National Committee for Clinical Laboratory Standards.
Paluszynski, J. P., Klassen, R., Rohe, M. & Meinhardt, F. (2006). Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae. Yeast 23, 707–715.[CrossRef][Medline]
Peters, G. J., Laurensse, E., Lankelma, J., Leyva, A. & Pinedo, H. M. (1984). Separation of several 5-fluorouracil metabolites in various melanoma cell lines. Evidence for the synthesis of 5-fluorouracil nucleotide sugars. Eur J Cancer Clin Oncol 20, 1425–1431.[CrossRef][Medline]
Polak, A. & Scholer, H. (1975). Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 21, 113–130.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Harbor, NY: Cold Spring Harbor Laboratory.
Sanglard, D., Ischer, F., Parkinson, T., Falconer, D. & Bille, J. (2003). Candida albicans mutations in the ergosterol biosynthesis pathway and resistance to several other agents. Antimicrob Agents Chemother 47, 2404–2412.
Schwarz, P., Dromer, F., Lortholary, O. & Dannaoui, E. (2003). In vitro interaction of flucytosine with conventional and new antifungals against Cryptococcus neoformans clinical isolates. Antimicrob Agents Chemother 47, 3361–3364.
Schwarz, P., Dromer, F., Lortholary, O. & Dannaoui, E. (2006). Efficacy of amphotericin B in combination with flucytosine against flucytosine-susceptible or flucytosine-resistant isolates of Cryptococcus neoformans during disseminated murine cryptococcosis. Antimicrob Agents Chemother 50, 113–120.
Schwarz, P., Janbon, G., Dromer, F., Lortholary, O. & Dannaoui, E. (2007). Combination of amphotericin B with flucytosine is active in vitro against flucytosine-resistant isolates of Cryptococcus neoformans. Antimicrob Agents Chemother 51, 383–385.
Smith, S. J., Crowley, J. H. & Parks, L. W. (1996). Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae. Mol Cell Biol 16, 5427–5432.[Abstract]
Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503–517.[CrossRef][Medline]
Te Dorsthorst, D. T. A., Verweij, P. E., Meletiadis, J., Bergervoet, M., Punt, N. C., Meis, J. F. G. M. & Mouton, J. W. (2002). In vitro interaction of flucytosine combined with amphotericin B or fluconazole against thirty-five yeast isolates determined by both the fractional inhibitory concentration index and the response surface approach. Antimicrob Agents Chemother 46, 2982–2989.
Vanden Bossche, H. (1985). Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action. In Current Topics in Medical Mycology, vol. 1, pp. 313–351. Edited by M. R. McGinnis. New York: Springer-Verlag.
Vanden Bossche, H., Willemsens, G. & Marichal, P. (1987). Anti-Candida drugs: the biochemical analysis for their activity. Crit Rev Microbiol 15, 57–72.[Medline]
Vanden Bossche, H., Marichal, P., Odds, F. C., Jeune, L. & Coene, M. C. (1992). Characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother 36, 2602–2610.
Vanden Bossche, H., Marichal, P. & Odds, F. C. (1994). Molecular mechanisms of drug resistance in fungi. Trends Microbiol 2, 393–400.[CrossRef][Medline]
Wach, A., Brachat, A., Pöhlmann, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.[CrossRef][Medline]
Wadler, S., Horowitz, R., Zhang, H. Y. & Schwartz, E. L. (1998). Effects of perturbations of pools of deoxyribonucleoside triphosphates on expression of ribonucleotide reductase, a G1/S transition state enzyme, in p53-mutated cells. Biochem Pharmacol 55, 1353–1360.[CrossRef][Medline]
Watson, P. F., Rose, M. E. & Kelly, S. L. (1988). Isolation and analysis of ketoconazole mutants of Saccharomyces cerevisiae. J Med Vet Mycol 26, 153–162.[Medline]
Watson, P. F., Rose, M. E., Ellis, S. W., England, H. & Kelly, S. L. (1989). Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole anti-fungals. Biochem Biophys Res Commun 164, 1170–1175.[CrossRef][Medline]
Whelan, W. L. (1987). The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans. Crit Rev Microbiol 15, 45–56.[Medline]
Yanisch-Perron, C., Viera, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Received 29 April 2008;
revised 12 June 2008;
accepted 19 June 2008.
This article has been cited by other articles:
|
|
 |
|
  B. A. McManus, G. P. Moran, J. A. Higgins, D. J. Sullivan, and D. C. Coleman A Ser29Leu Substitution in the Cytosine Deaminase Fca1p Is Responsible for Clade-Specific Flucytosine Resistance in Candida dubliniensis Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4678 - 4685. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
), FLC (
) and 5FC+FLC (
). (a) S. cerevisiae wild-type BY4741, (b) 
