Flucytosine (5-FC) belongs to the class of pyrimidine ana- logues and was developed in the 1950s as a potential antine- oplastic agent. Abandoned as anti-cancer drug due to its lack of activity against tumors, it showed, however, good in vitro and in vivo antifungal activity. Because it is highly water- soluble, it can be administered by either oral or intravenous routes [19]. 5-FC is taken up by fungal cells by a cytosine permease and is deaminated by a cytosine deaminase to 5-fluorouracil (5-FU), a potent antimetabolite. 5-FU can be converted to a nucleoside triphosphate and, when incorpo- rated into RNA, causes miscoding. In addition, 5-FU can be converted to a deoxynucleoside, which inhibits thymidilate synthase and thereby DNA synthesis. 5-FC shows little tox- icity in mammalian cells, because cytosine deaminase is absent or poorly active in these cells. 5-FU is a potent anti- cancer agent, but it is impermeable to fungal cells. The con- version of 5-FC to 5-FU by intestinal bacteria is possible, and therefore 5-FC can show toxicity in oral formulations.
5-FC is fungicidal for susceptible fungi. High variability in 5-FC MICs is observed in Candida species and C. neofor- mans because of the occurrence of intrinsic resistance. MIC90 of 5-FC are in the range of 0.5–4 mg/mL for Candida spe- cies, including C. albicans, C. parapsilosis, C. tropicalis and C. glabrata, and for C. neoformans [20].
5-FC is not usually administered as a single agent because of rapid development of resistance. It is therefore used mainly in combination with other agents, particularly AmB. In vitro
data regarding the combination of both drugs against Candida species and C. neoformans are numerous and are contradic- tory, showing antagonistic, indifferent or synergistic effects [21]. 5-FC is an antifungal agent against which resistance can be intrinsic or acquired. Resistance may occur due to the deficiency or lack of enzymes implicated in the metabolism of 5-FC, or resistance may be due to the deregulation of the pyrimidine biosynthetic pathway, in which products can compete with the fluorinated metabolites of 5-FC. Detailed investigations on the molecular mechanisms of resistance to 5-FC have shown that intrinsic resistance to 5-FC in fungi can be due to a defect in the cytosine permease, as observed in C. glabrata, but not in C. albicans or C. neoformans.
Acquired resistance results from a failure to metabolize 5-FC to 5-FUTP (5-fluorouridine triphosphate) and 5-FdUMP (5-fluoro-deoxyuridine monophosphate), or from the loss of feedback control of pyrimidine biosynthesis [22].
Recently, a mutation in the FUR1 gene encoding uracil phosphoribosyltransferase was reported as a cause of 5-FC resistance. This mutation (cytosine to thymine at position 301) is thought to decrease the conversion of 5-FU, which is produced from deamination of 5-FC, into a toxic metabo- lite (5-fluorouridine monophosphate) and thus counteracts the action of this compound [23]. Interestingly, this resis- tance mechanism is enriched in a C. albicans subpopulation (clade I), which is one among the five major clades (I, II, III, SA, and E) known in this yeast species. In this population, the FUR1 mutation can occur in the hetero- or homozygous state and is correlated with increased 5-FC MIC value. In addition to this specific mechanism, a mutation in cytosine deaminase (FCA1) has been reported correlating with 5-FC resistance in C. albicans [24].
Azoles
Azole antifungal agents discovered in the late 1960s are synthetic compounds belonging to the largest group of anti- fungal agents. Azole antifungal agents used in medicine are categorized into N-1 substituted imidazoles (ketoconazole, miconazole, clotrimazole) and triazoles (fluconazole, itra- conazole, posaconazole, voriconazole, and ravuconazole (Fig. 2). Azoles have a cytochrome P450 as a common cel- lular target in yeast or moulds (Fig. 3). This cytochrome P450, now referred to as Erg11p, is the product of the ERG11 gene. The unhindered nitrogen of the imidazole or triazole ring of azole antifungal agents binds to the heme iron of Erg11p as a sixth ligand, thus inhibiting the enzymatic reac- tion. The affinity of imidazole and triazole derivatives is not only dependent on this interaction, but is also determined by the N-1 substituent, which is actually responsible for the high affinity of azole antifungal agents to their target. Each
138 D. Sanglard
of these agents has distinct pharmacokinetics and their antifungal efficacies are quite different among yeast and moulds of medical relevance.
Azole antifungals have a broad spectrum of activity. They are active against Candida species, C. neoformans and dimorphic fungi. Some azole derivatives are more active than others for different fungi. For example, fluconazole is rela- tively inactive against C. krusei or A. fumigatus as compared with itraconazole or voriconazole. Posaconazole shows high activity against a large number of filamentous fungi and has a more predictable pharmacodynamic profile than voricon- azole; it is also active against fungal pathogens, such as the zygomycetes, which are refractory to other azoles [25].
Azole antifungals are only fungistatic against most yeast species. Against Candida infections, fluconazole has demon- strated broad clinical efficacy for mucosal candidiasis, Candida urinary tract infections, candidemia, and invasive candidiasis [26].
Reports on resistance to azole antifungal agents were rare until the late 1980s. The first cases of resistance were reported in C. albicans after prolonged therapy with micon- azole and ketoconazole. Following the use of fluconazole for a wide variety of infections, especially the treatment of mucosal infections in AIDS patients, antifungal resistance to this agent has been more frequent [27]. There are several
mechanisms by which yeasts or filamentous fungi can become resistant to azole antifungal agents (Fig. 3).
Resistance by Altered Drug Transport
Failure to accumulate azole antifungals has been identified as a cause of azole resistance in several post-treatment clinical fungal isolates. These isolates include C. albicans, C.
glabrata, C. krusei, C. dubliniensis and C. neoformans [28].
In azole-resistant yeasts, genes encoding ATP Binding Cassette (ABC)-transporters were upregulated as compared to the corresponding azole-susceptible species. So far, only CDR1 (Candida Drug Resistance 1) and CDR2 in C. albicans [29, 30], CdCDR1 in C. dubliniensis [31, 32], a CDR1 homo- logue in C. tropicalis [33], CgCDR1, CgCDR2 (also known as PDH1) and CgSNQ2 in C. glabrata [34–36] and AFR1 in C. neoformans [37] have been identified as ABC-transporter genes upregulated in azole-resistant isolates.
In A. fumigatus, itraconazole is able to induce an ABC- transporter gene, atrF; however, the role of this gene in the resistance of clinical isolates to itraconazole is still not estab- lished in detail [38]. Heterologous expression of ABC- transporter genes such as CDR1 and CDR2, CdCDR1,
Fig. 2 Chemical structures of major antifungal agents
139 Resistance to Antifungal Drugs
CgCDR1 and CgCDR2 in S. cerevisiae conferred resistance not only to several azole derivatives (fluconazole, itracon- azole, ketoconazole) but also to a wide range of compounds, including antifungals and metabolic inhibitors [30, 35, 39].
Several laboratories have also observed that, besides upregulation of ABC-transporter genes, a multidrug trans- porter gene named MDR1 (Multidrug Resistance 1), also pre- viously known as BEN (Benomyl resistance) and belonging
Fig. 3 Schematic view of the four mains resistance mechanisms to azole antifungals in yeast pathogens. Erg11p, the cellular target of azole antifungals, is responsible for the demethylation of lanosterol.
14a-demethlylated sterols serve as further substrates in the formation of ergosterol. When azole drugs bind Erg11p, lanosterol demethylation
is blocked and sterol metabolites remains methylated at the position 14a. The toxic metabolite 3,6-diol (14a-methylergosta-8,24(28)-dien- 3b,6a-diol) is formed from the action of ERG3 on 14a-methylfecosterol (see Fig. 2 for sterol structures). Details on specific resistance mecha- nisms are given in the text
140 D. Sanglard
to the family of Major Facilitators, was upregulated in some C. albicans azole-resistant clinical isolates. Deletion of MDR1 in C. albicans and also in C. dubliniensis isolates with acquired azole resistance by MDR1 upregulation resulted in a sharp increase of azole susceptibility, thus sup- porting by this genetic approach, the involvement of this spe- cific gene in azole resistance [40, 41]. Deletion of MDR1 in an azole-susceptible laboratory strain did not result in a sig- nificant increase of azole susceptibility, thus supporting the fact that MDR1 is almost not expressed in this type of strain and more generally in azole-susceptible clinical isolates [42].
Upregulation of an MDR1-like gene has been also observed in a fluconazole-exposed C. tropicalis isolate that acquired cross-resistance to fluconazole and itraconazole [33].
In most cases, azole resistance acquired in clinical situa- tions by yeast pathogens by multidrug transporters is main- tained over a high number of generations in vitro without drug selection. Azole resistance can however be a reversible phenomenon. Marr and collaborators [43] obtained C. albi- cans isolates that developed azole resistance from bone marrow transplant patients being treated with fluconazole.
An increase in the fluconazole MIC was coupled with upreg- ulation of CDR1, but was decreased with a parallel decrease in CDR1 expression in drug-free subculture. Azole- susceptible isolates from this type of patient, when exposed in vitro to fluconazole, developed reversible azole resistance by the same CDR1 upregulation mechanism. Interestingly, only a portion of individually exposed colonies were ren- dered less susceptible to fluconazole, thus indicating that hetero-resistance, which was already described in azole- exposed C. neoformans isolates, could occur in specific C.
albicans isolates [44].
Another interesting acquisition of azole resistance by multidrug transporter upregulation in a clinical context has been shown in C. glabrata. This yeast could convert to azole resistance by loss of mitochondrial DNA. The phenomenon, also called HFAR, for High Frequency Azole Resistance, because it occurred in vitro at high frequencies, was coupled with upregulation of CgCDR1 and CgCDR2 [34].
Interestingly, mitochondrial defects have been reported in several clinical isolates that were exposed to fluconazole during treatment [45, 46].
The molecular basis for the upregulation of multidrug transporters belonging to the ABC and Major Facilitator families has been actively investigated in yeast pathogens.
It is believed that the mutation(s) leading to gene upregula- tion might rather be caused by alterations involving tran- scription factors. Using the Renilla luciferase reporter system fused to CDR1 and CDR2 promoters cloned from azole-susceptible isolates, De Micheli et al. [47] showed that their expression was enhanced in an azole-resistant strain, in which these genes are constitutively upregulated.
With another reporter system, GFP fused to the MDR1
promoter from an azole-susceptible strain, Wirsching et al.
[48] showed that high fluorescence could be obtained when the chimeric construct was introduced in an azole-resistant strain upregulating MDR1.
The systematic dissection of the CDR1 and CDR2 pro- moters allowed the identification of five distinct regulatory elements: the basal expression element (BEE) responsible for basal expression; the drug-responsive element (DRE) required for the response to drugs, such as fluphenazine and estradiol; two steroid responsive elements (SRE) involved in the response to steroid hormones; and the negative regu- latory element (NRE) [47, 49, 50]. Internal deletions of the BEE and DRE in the CDR1 promoter affect basal CDR1 expression and drug-induced expression, respectively.
Conversely, the deletion of the NRE leads to an increase in the basal expression of CDR1. In contrast to CDR1, the CDR2 promoter only contains the DRE element [47].
Among these different cis-acting elements, only the DRE was shown to be involved not only in the transient up-regu- lation of both CDR1 and CDR2 in response to inducers but also in their constitutive high expression in azole-resistant clinical isolates [47].
The DREs present in the promoter of CDR genes contain two CGG triplets which are potentially recognized by Zn2- Cys6 transcription factors [51–54]. In order to isolate regula- tors of CDR1 and CDR2, the C. albicans genome was searched for genes encoding proteins with Zn2-Cys6 fingers.
Interestingly, three of these genes were arranged in tandem near the mating locus (MTL), the homozygosity of which is linked to the development of azole resistance in C. albicans [55]. Deletion of one of these genes, TAC1 (transcriptional activator of CDR genes), in an azole-susceptible strain led to increased drug susceptibility and to loss of transient CDR1 and CDR2 upregulation in presence of inducers. In C. albi- cans clinical isolates resistant to azoles, deletion of TAC1 abolishes CDR1 and CDR2 expression and therefore, drug resistance, demonstrating that TAC1 is the main mediator of azole resistance due to the upregulation of ABC transporter in C. albicans [56]. Tac1p acts by direct binding to the DRE present in the promoter region of both efflux pump genes and induces their expression in response to steroid and several toxic chemicals [47, 56]. Tac1p is not involved in the basal expression of CDR1 and the transcription factor regulating CDR1 expression through the BEE element remains to be identified.
Functional dissection studies of the MDR1 promoter have identified two distinct regulatory regions. The benomyl response element (BRE), also called the MDR1 drug resis- tance element (MDRE) is responsible for the constitutive high expression of MDR1 in fluconazole-resistant isolates [57–59]. This region is also necessary for the inducible expression of MDR1 in response to benomyl [57, 59]. The second element, the HRE (H2O2 response element) is involved
141 Resistance to Antifungal Drugs
in the response to oxidative stress agents. In contrast to the BRE, the HRE is not required for constitutive upregulation of MDR1 in azole-resistant isolates. The HRE region con- tains two YRE (YAP1 response element) motifs and the BRE/MDRE contains a perfect match for the Mads-box tran- scription factor Mcm1p [58, 60, 61]. However there is no direct evidence either for interactions between Yap1p and the HRE and between Mcm1p and the BRE or for the involve- ment of Yap1p and Mcm1p in the inducible or constitutive expression of MDR1.
The molecular basis for the constitutive upregulation of the Major facilitator gene MDR1 has been recently eluci- dated. A genome-wide study was undertaken to compare the transcriptional profiles of three different C. albicans clinical isolates overexpressing MDR1 in order to identify genes commonly upregulated with MDR1. One of the genes of interest was orf19.7372 since it contained a Zn2-Cys6 zinc finger motif of the same type as TAC1. Because inactivation of orf19.7372 caused loss of MDR1 upregulation, the tran- scription factor was called Mrr1pp (multi-drug resistance regulator) [62]. MRR1 inactivation in azole-resistant isolates resulted in the loss of MDR1 expression and increased sus- ceptibility to fluconazole, cerulenin and brefeldin A [62].
Deletion of MRR1 in a drug-susceptible strain abolished MDR1 upregulation in presence of inducing chemicals, such as benomyl and H2O2, thus demonstrating that Mrr1p medi- ates both inducible MDR1 expression and constitutive MDR1 overexpression in drug-resistant strains [62]. A MRR1 homolog has been identified in C. dubliniensis and was shown responsible for CdMDR1 expression [63]. Although Mrr1p has not been yet shown to bind directly to the MDR1 promoter in order to regulate MDR1 expression, CdMrr1p is able to activate the MDR1 promoter in the heterologous spe- cies C. albicans, suggesting that Mrr1p and CdMrr1p recog- nize the same binding site(s) which should be present in the MDR1 promoters of both species [63].
The identification of trans-acting factors regulating ABC- transporters in pathogenic fungi can also rely on the well- described Pleiotropic Drug Resistance (PDR) network involved in multidrug resistance in S. cerevisiae. The two Zn2-Cys6 transcription factors PDR1/PDR3 are master regu- lators of this network and control multidrug resistance by modifying the expression of several ABC (PDR5, SNQ2 and YOR1)- and MFS (FLR1, TPO1)-transporters as well as other genes determining cell membrane composition (PDR16, RSB1, LPT1). In silico search for PDR1/PDR3 homologues in the genomes of pathogenic fungi has been performed, and so far only one functional homolog, CgPDR1, was described in C. glabrata [64]. CgPdr1p has 40% and 35% identity with Pdr1p and Pdr3p, respectively, and could complement a pdr1D S. cerevisiae mutant strain [65]. Similar to S. cerevi- siae, the expression of C. glabrata CgCDR1/2 and CgSNQ2 genes is regulated by CgPdr1p [64]. Deletion of CgPDR1 in
C. glabrata azole-resistant clinical isolates leads to a loss of CgCDR1, CgCDR2 and CgSNQ2 regulation and to a sharp increase in azole susceptibility, indicating that CgPDR1 is the main regulator of efflux-mediated azole-resistance in C.
glabrata [36, 66]. Deletion of CgPDR1 also abolishes CgCDR1 and CgCDR2 up-regulation in presence of flucon- azole [66].
It is now well established that S. cerevisiae Pdr1p and Pdr3p act through cis-acting sites present in the promoters of target genes. The consensus motif is named PDRE (for pleio- tropic drug resistance element) and is present in several ABC-transporter gene promoters such as PDR5, SNQ2 and YOR1 [67]. In C. glabrata, a genome-wide study identified genes regulated by CgPDR1 and, by analysis of the promot- ers, the sequence 5¢-TCC(GA)(CT)GAA-3¢ was identified as a strong candidate for C. glabrata PDRE. This sequence is found in the promoters of CgCDR1, CgCDR2 and CgSNQ2 genes, suggesting that CgPdr1p binds directly to PDRE sequences to regulate transcription of target genes [36, 65, 66].
CgPDR1 contains a PDRE in its promoter suggesting an auto-regulation of its transcription. Consistent with this observation, upregulation of CgCDR1 and CgCDR2 is cor- related in some azole resistant strains with an increase of CgPDR1 expression [65, 66]. However, although promoters regions of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1 genes all contain PDRE, they are not simultaneously expressed in clinical azole-resistant isolates [45]. CgPdr1p acts as nuclear receptor by directly binding to diverse drugs and xenobiot- ics, such as azoles, to activate expression of efflux pumps genes resulting in multidrug resistance [68]. A small portion of the activation domain of CgPdr1p binds directly to the KIX domain of the Mediator co-activator subunit CgGal11p in a xenobiotic-dependent manner in order to activate tran- scription of target genes [68].
One of the consequences of the involvement of multidrug efflux transporters in resistance to azole antifungals is that these transporters have the ability to mediate cross-resistance to unrelated antifungals or metabolic inhibitors. In order to determine whether or not a given substance is a potential substrate for multidrug efflux transporters, different approaches have been taken. One consists of functional expression of the C. albicans multidrug efflux transporters in the baker’s yeast S. cerevisiae carrying a deletion of the PDR5 gene [30, 42]. Depending on the acquisition of resis- tance of S. cerevisiae mutants expressing these specific trans- porters against a given compound, the substance can be considered as a potential substrate for the expressed multi- drug transporter. Potential substrates for the multidrug efflux transporters encoded by CDR1 and CDR2 include almost all azole antifungals of medical importance, including flucon- azole, itraconazole, posaconazole, and voriconazole, and other antifungal agents, such as terbinafine and amorolfine.
For the multidrug transporter encoded by MDR1, fluconazole
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was the only relevant substrate among azole antifungals.
Several antifungal agents could not be assigned as substrates (AmB, 5-fluorocytosine). Since not only azoles, but other antifungals, such as terbinafine and amorolfine, can be taken simultaneously as substrates by several multidrug efflux transporters, multidrug efflux transporter genes, when over- expressed in clinical yeast isolates, have the potential of mediating cross-resistance to different antifungal agents.
Data suggest that the upregulation of MDR1 is responsible for the specific resistance to fluconazole in Candida isolates, and this is consistent with the observation that MDR1 over- expression in S. cerevisiae was only conferring resistance to fluconazole [30].
Resistance to Azole Antifungals Involving Alterations of the Cellular Target
Target alterations are known resistance mechanisms for two classes of antifungal agents, azoles and echinocandins.
Resistance mediated by alterations in Erg11/Cyp51, the tar- gets of azoles, has been widely documented involving either mutations or upregulation of their genes. A large number of non-synonymous nucleotide polymorphisms (up to 110, including 100 with unique substitutions) have been described in ERG11 alleles originating from C. albicans azole-resistant isolates. The degree of ERG11 polymorphism is therefore high and suggests that Erg11p is highly permissive to struc- tural changes resulting from amino acid substitutions. The contribution of each individual mutation to azole resistance is, however, difficult to estimate because ERG11 mutations often occur in combination (from 2 to 4 combined mutations) in the same allele and because resistance mechanisms are often combined in azole-resistant C. albicans isolates [69].
Using different approaches, including heterologous expres- sion in S. cerevisiae, enzyme assay in C. albicans extracts, and site directed mutagenesis, evidence for their involvement in azole resistance has been provided for at least some of these mutations (F72L, F145L, G464S, Y132F, R467K, S405F) [70–76].
In A. fumigatus, itraconazole resistance in clinical isolates is associated with the occurrence of amino acid substitution in Cyp51A, which is the functional ortholog of Erg11p in this fungal species. Interestingly, mutations at position G54 con- tribute only to itraconazole resistance and not to voriconazole resistance [77, 78]. In contrast, mutations at position M220 confer itraconazole resistance and also high MICs to voricon- azole and posaconazole [79]. Similarly, mutations at positions L98 and G138 recently described in Cyp51A conferred cross-resistance to all azoles [79]. The Cyp51A mutation L98H is consistently combined with cyp51A upregulation.
This mechanism allows cross-resistance to all known azoles [80]. Intriguingly, the L98H substitution and cyp51A upregu- lation mechanisms were also found in isolates of environ- mental origin, thus raising the question on how azole resistance was acquired in a non-medical environment [81].
In C. neoformans, analysis of ERG11 from a clinical azole-resistant isolate showed a point mutation linked an amino acid substitution G484S that was not observed in the parent azole-susceptible isolate [82]. Recent studies demon- strated that azole resistance in this yeast species can be due to heteroresistance, which is a mechanism by which resis- tance can be induced or reversed in a portion of a growing population [83].
Upregulation of ERG11 has been mentioned as a possible cause of azole resistance in a few cases in C. albicans, C.
glabrata clinical isolates and in a single C. tropicalis isolate [84]. Upregulation of ERG11 does not exceed a factor of 3–5 in azole-resistant isolates when compared to ERG11 expression in related azole-susceptible strains [29, 85–87]. Upregulation of ERG11 can be achieved in principle by deregulating gene transcription or by gene amplification.
In S. cerevisiae, ERG11 is regulated by two transcrip- tional activators, Upc2p and Ecm22p, which are members of the Zn2-Cys6 transcription factor family [88]. They act through binding to regulatory elements present in the ERG11 promoter called the Sterol Regulatory Element (SRE). Other SREs are found in genes involved in sterol biosynthesis. A single C. albicans gene (UPC2) with homology to both S.
cerevisiae genes, has been identified and characterized [89, 90]. Deletion of UPC2 in C. albicans caused loss of ERG11 upregulation in response to azole drugs, which occurs other- wise in the parent strain. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from −224 to −251 upstream of the start codon.
This segment contains two 7-bp sequences (5¢ TCGTATA 3¢) separated by 13-bp [91] forming an imperfect inverted repeat, a typical feature for binding to Zn2-Cys6 transcription factors [67]. The Upc2p core binding sequence is conserved between Candida and Saccharomyces. This core is found in the ERG11 promoter in a region identified as important for azole induction of ERG11 expression [90].
As mentioned above, upregulation of cyp51A in A. fumig- atus has been detected in clinical isolates with cross-resis- tance to several azole antifungal agents. This upregulation is associated with a (L98H) substitution in Cyp51A and with the presence of a 34-bp tandem repeat in the cyp51A pro- moter [80]. This resistance mechanism has been also identi- fied in A. fumigatus isolates originating from the environment in the Netherlands. Exposure of environmental isolates to agricultural azole fungicides is suspected as a possible cause of the emergence of such azole-resistant isolates recovered from treated patients [81].