The cellular and molecular defense mechanisms of the Candida yeasts against azole antifungal drugs

Journal de Mycologie Médicale (2012) 22, 173—178

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GENERAL REVIEW/REVUE GE´NE´RALE

The cellular and molecular defense mechanisms of the Candida yeasts against azole antifungal drugs ´ canismes cellulaires et mole ´ culaires de de ´ fense des Candida contre Les me ´s les antifongiques azole T. Noël ´ de Bordeaux, CNRS, Microbiologie fondamentale et Pathoge ´ nicite ´ , UMR 5234, 33000 Bordeaux, France Universite Received 9 April 2012; accepted 13 April 2012 Available online 23 May 2012

KEYWORDS Azole antifungals drugs; Candida; Resistance; Mutation; Upregulation; Genome plasticity

MOTS CLÉS Antifongiques azolés ; Candida ; Résistance ; Mutation ; Surexpression ; Plasticité du génome

Summary The molecular mechanisms supporting resistance to azole antifungals have attracted a great interest during the last decades because of the emergence of clinical resistance to the treatment of fungal infections. The availability of genome sequencing data, of molecular biology tools, and of a large set of clinical and laboratory azole-resistant strains, made the yeasts Candida the biological material of choice to decipher azole resistance mechanisms. The yeast Candida albicans has several cellular ways to resist to azole drugs: decreased affinity of the target protein Erg11p for the drugs, increased biosynthesis of Erg11p, and efflux of the drugs outside the fungal cells. At the molecular level, two main mechanisms are operating: point mutation in the target gene or in transcriptional activator factors, eventually associated to a loss of heterozygosity, and gene duplication that results from the extraordinary plasticity of the genome. This review proposes to explore the different molecular strategies that are used by Candida yeasts to fight azole antifungals. # 2012 Published by Elsevier Masson SAS. Résumé Les mécanismes moléculaires de résistance aux antifongiques azolés ont un grand intérêt au cours de ces dernières années à cause de l’émergence de résistances cliniques aux traitements des infections fongiques. La disponibilité des données sur du séquençage des génomes, d’outils moléculaires et d’abondantes séries de souches cliniques et de laboratoire azole-résistantes, ont fait des Candida un matériel biologique de choix pour élucider les mécanismes de résistance aux antifongiques azolés. C. albicans possède différentes mécanismes cellulaires pour résister aux azolés : diminution de l’affinité de la cible protéique Erg11p pour les antifongiques, augmentation de la biosynthèse de la protéine Erg11p et efflux des antifongiques hors de la cellule. Au niveau moléculaire, deux mécanismes sont opérationnels : mutations ponctuelles du gène cible ou des facteurs activateurs de transcription, éventuellement associées à une perte de l’hétérozygotie, et duplication de gènes qui résulte de l’extraordinaire plasticité

E-mail address: [email protected]. 1156-5233/$ — see front matter # 2012 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.mycmed.2012.04.004

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T. Noël du génome. Cette revue propose d’explorer les différentes stratégies moléculaires utilisées par les Candida pour lutter contre les antifongiques azolés. # 2012 Publié par Elsevier Masson SAS.

Azole antifungal molecules have been and are still widely used to treat both superficial mucosal and deep and disseminated fungal infections. However, extensive use favored the development of resistance and resulted in therapeutic failure. Historically, concerns with azole resistance started in the mid-1980s with the treatment of the oropharyngeal candidiasis of the patients VIH+ using fluconazole. Inadequate posology resulting in under-dosing, combined to the fact that fluconazole was fungistatic, contributed largely to the selection of clinical resistant isolates, notably of Candida albicans [34]. However, a strong mobilization of the scientific community allowed elucidation of the molecular mechanisms underlying fluconazole resistance in the yeasts Candida, of which a number of them could be extrapolated to the entire triazole family. By considering both their frequency and their efficiency, there are three main mechanisms leading to azole resistance [35]:

Once bound to the catalytic pocket of the enzyme, azole antifungals prevent demethylation of lanosterol. This leads to a deficit of ergosterol, and thus has multiple consequences onto the yeast cell physiology. Indeed, ergosterol plays important roles in the fungal cells such as the correct functioning of membrane proteins, membrane compartmentalization, endocytosis, vacuole fusion and pheromone signaling, as mentioned in a recent work which demonstrates that the simple binding of ergosterol by another antifungal molecule, amphotericin B, is sufficient to kill a yeast cell [9]. Another consequence of the action of azoles is the accumulation of methylated sterols which are toxic for the fungal cell, notably 14a-methyl-ergosta-8,24(28)-dien-3b,6a-diol, more simply called 14a-methyl-3,6-diol [14].

 point mutations of ERG11, the gene encoding the target protein of azole, that reduce the binding affinity of the protein to azole antifungals;  overexpression of ERG11, leading to an increase of the intracellular concentration of the target protein;  overexpression of efflux membrane transporters, which decreases the intracellular concentration of the antifungal drug.

In C. albicans, a single gene of 1587 bp encodes the 595 amino acids Erg11p. Nearly 150 non synonymous point mutations have been identified hitherto in clinical resistant isolates of C. albicans [19]. These mutations are not randomly distributed over the ERG11 gene, and are preferentially clustered within three hot spot regions. One of them (amino acid 105 to 165) encompasses the N-ter helix-rich segments of the protein forming a channel for allowing the substrates, and possibly the antifungals, to reach the catalytic pocket. The two other hot spots cover regions of the protein (aa 266 to 287, and aa 405 to 488) that are important for the fixation of the heme [18]. Amino acid substitutions are thought to decrease the global binding affinity of the protein to the antifungal, either by hampering the access of the drug to the heme, or by preventing the coordination of the drug to the heme. However, many of the mutations identified have no incidence on resistance and only participate to the natural genetic polymorphism occurring in any living organism. Few of them were demonstrated to really support azole resistance. Generally, an univoqual demonstration is provided by performing the allelic replacement in a yeast strain of a wild ERG11 allele by a mutated allele and measuring the effect of the replacement on the Minimal Inhibitory Concentration (MIC) of the antifungal molecule. Generally, allelic replacement is carried out in a laboratory model yeast such as Saccharomyces cerevisiae, but other yeast species, provide the fact they are haploid and easily amenable to DNA-based genetic manipulations, are often preferred because S. cerevisiae shows naturally high MICs to fluconazole [2]. Alternative biochemical methods can be used to test the effect of a mutation. They consist in comparing the ratio of the cellular content of the methylated sterol versus ergosterol in resistant and susceptible isolates [11], or assaying the kinetic parameters (Km, Vmax) of the Erg11 protein in the presence of radiolabeled 14a-methyl sterol substrates [15]. Lastly, an in silico method relying on computed three dimensional models of Erg11p can be used to evaluate the probability that a particular amino acid substitution in Erg11p has a

The target of azole antifungals and mechanism of inhibition In the yeasts Candida, the target of azole antifungals is the protein Erg11p. This protein of about 60 kDa is located to the inner face of the membrane of the smooth endoplasmic reticulum, an organelle which is involved in a number of biological pathway, notably in the synthesis of lipids, including oils, phospholipids and steroids. Precisely, Erg11p is involved in the biosynthesis of ergosterol, the main sterol of the membranes of the fungal cells. Erg11p is a lanosterol 14-alpha-demethylase; it catalyzes the C-14 demethylation of lanosterol to form 4,400 -dimethyl cholesta-8,14,24-triene-3-beta-ol. Member of the cytochrome P450 family, Erg11p is a hemoprotein that has an iron atom as prosthetic group, whose function is to fix the dioxygen necessary for the enzymatic activity. Indeed, the enzyme needs to oxidize three times successively the substrate lanosterol to progressively eliminate the methyl group as formic acid [30]. To accomplish its function, Erg11p requires also protons and thus makes part of a complex of oxido-reductases; it is associated and coordinately regulated with the NADPH-cytochrome P450 reductase Ncp1p, which acts as the donor of electron [33]. Azole antifungals behave as competitive inhibitors of O2 fixation by binding the sixth coordination site of the heme iron by the N-heterocycle nitrogen of the triazole ring [22].

Point mutations of ERG11

Mechanisms of azole resistance in Candida yeasts

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chance to decrease the binding affinity to the antifungal molecule. However, the bioinformatics 3D models available are all derived from the soluble CYP51 protein of Mycobacterium tuberculosis (Mt), whose crystal structure was resolved in complex with fluconazole [22]. Eukaryotic orthologs, such as the fungal Erg11 proteins, are more difficult to crystallize because tightly attached by a 30 aa N-ter transmembrane helix to microsomal and smooth endoplasmic reticulum membrane fractions [37]. The Mt model allowed validation of a certain number of aa substitution involved in azole resistance. For example, the impact of mutations resulting in the substitutions K143R, T315A, S405F, G464S, R467K or I471T was clearly demonstrated by allelic replacement and was confirmed by 3D models [e.g. 15; for a review 19]. However, bacterial and fungal Erg11p are quite different, as they exhibit only 30% amino acid identity. Furthermore, the fungal Erg11p possesses an internal extrapeptidic loop of 15 to 19 residues (depending on the fungal species) in the C-ter region of the protein, also called the P-loop, which was not modeled in detail [37]. Recently, it was shown that some amino acid substitutions in the P-loop could confer azole-resistance, and a new model was proposed for the fungal Erg11p [1]. In vitro modeling also served for docking studies, i.e. the computational investigation of the possible binding modes of an azole inhibitor to its target protein Erg11p. It is necessary to distinguish two subgroups among the triazole antifungals: itraconazole and posaconazole which both possess a long side chain that makes extensive hydrophobic contacts along its entire length with the fungal enzyme, and fluconazole and voriconazole, whose side chain is only constituted by a single heterocycle, and which have much less interaction with Erg11p. Docking studies support the idea that posaconazole is more strongly bound to its target than do fluconazole and voriconazole, and therefore that some mutations of the ERG11 gene and amino acid substitutions could confer resistance to fluconazole and voriconazole, and not necessarily to posaconazole [37]. This hypothesis seems to be confirmed to some extent by the data collected from clinical observations [21]. Overall, mutations in ERG11 are responsible for variable azole MIC increases depending on the position of the mutation. In the case of fluconazole MIC, from 2- to 64-fold increase were observed [19].

acids homologue to the S. cerevisiae Upc2p and Ecm22p was identified and characterized in C. albicans [31,16]. This protein, named Upc2p, is a member of a family of transcription factors that are specific to fungi, the Zn(II)2-Cys6 binuclear cluster family. The N-ter part of Upc2p contains six cystein residues binding to two zinc atoms which coordinate folding of the DNA-binding domain. Once bound to the specific SRE box (Sterol Response Element) located in the promoters of different ERG genes, an activation domain, present at the C-ter portion of the protein, is able to activate the transcription of the gene. Electrophoretic mobility shift assay (EMSA) was used to demonstrate that Upc2p was able to bind the SRE box of ERG2, encoding C-8 sterol isomerase, suggesting that all the ergosterol biosynthetic genes which have a SRE box in the promoter, like ERG11, are directly regulated by Upc2p [16]. The UPC2 gene expression is induced in the presence of antifungal drugs, including fluconazole, and Upc2p upregulates the expression of the ERG2 and ERG11 genes when C. albicans is grown under azole drug pressure [31,16]. Candida yeasts are thus able to sense the presence of azole antifungals, maybe indirectly through the decrease of ergosterol content, and to adapt the level of expression of the ERG biosynthetic pathway owing to Upc2p. Different strains of C. albicans, and probably of other Candida species, can express different alleles of UPC2 encoding transcriptional factors of different strength. These last years, the research focused on the identification of mutations of UPC2 with the idea to characterize transcription factors having a constitutive enhanced activity, and thus conferring loss of susceptibility to azole antifungals. Three mutations (A643T, A643V, G648D), so called mutations gainof-function, were characterized in sequential clinical isolates overexpressing ERG11, and were confirmed by allelic replacement in laboratory strains. They are all located in the C-ter part of the transcription factor, near the activation domain [13,7,10]. However, it should be noted that the contribution of mutations in UPC2 to azole resistance is weak: in one study, the mutated Upc2p[A643V] allowed a 20-fold increase of ERG11 mRNA, but only a two-fold increase in fluconazole MIC, from 2 mg/ml to 4 mg/ml [13]. Such a discrepancy may be explained by the fact that an accumulation of mRNA in the cell does not necessarily mean that they are all translated in protein.

Overexpression of ERG11

Upregulation of drug efflux pumps

Upregulation and overexpression of the ERG11 gene were suspected for a long time to be responsible for the loss of susceptibility to azole antifungals. Accordingly, different methods were used to measure the level of expression of ERG11 through the detection and quantification of its mRNA, such as Northern blot hybridization, semi-quantitative reverse transcriptase (RT) PCR, real-time quantitative RTPCR, and microarray analysis [24]. From 3 to 20-fold increase in mRNA could be observed according to the studies (e.g. x 3.7 in [17]; x 20 in [13]). Two independent mechanisms were shown to drive ERG11 overexpression. The first one is linked to a ERG11 gene duplication, as demonstrated for the first time in C. glabrata [17]. This mechanism is more detailed in a next paragraph. The second one depends on the regulation of the ergosterol biosynthesis pathway. A protein of 712 amino

The third mechanism relies on membrane transporter proteins whose activation results in the efflux of azole antifungals outside the cell, thus decreasing the intracellular concentration of the inhibitor. Two families of drug efflux transporters can be distinguished in yeasts, depending on the source of energy used for extrusion of the substrates, and on their spectra for the different azole molecules. The ATPBinding cassette (ABC) transporters, encoded by the genes CDR1 and CDR2 in Candida albicans [23,25], are constituted by two transmembrane domains, each consisting of six transmembrane segments, and by two cytoplasmic sites for the binding and hydrolysis of ATP dedicated to furnish energy. The substrate spectrum of the Cdr pumps is large: normally involved in phospholipids and steroid transport [32], they are able to transport all azole antifungals (and

176 also other toxic molecules such as cycloheximide for example), and therefore to promote cross-resistance to all azoles when activated. The Major Facilitator Superfamily (MFS) transporter is encoded by a single gene MDR1 in C. albicans. The protein contains twelve transmembrane spans [8] and utilizes a proton electrochemical gradient as source of energy for the transport of phospholipids (regular function) and drugs when present. Among azole substrates, Mdr1p is specific to fluconazole [36], and can also export other toxics, like cycloheximide or methotrexate. A study of the single-nucleotide polymorphism (SNP) of CDR1 and CDR2 in genetically unrelated strains of C. albicans revealed that the CDR1 gene, which is more often involved in azole efflux than CDR2, has very few non synonymous SNP, while the CDR2 exhibits higher polymorphism [12]. However, when tested by allelic replacement in a heterologous S. cerevisiae laboratory model, SNPs in CDR genes do not significantly contribute to variations in azole resistance [12]. This was confirmed by a study of the polymorphism of the promoter region of the MDR1 gene and of its possible incidence on the level of fluconazole resistance. Of two MDR1 alleles exhibiting one SNP in the promoter region, one allele had an increased expression level, but it was not sufficient to be correlated to an increase in fluconazole MIC [3]. In drug susceptible Candida strains, Cdr and Mdr transporters are expressed at a level compatible with their normal physiological role, i.e. phospholipids export. Drug resistant isolates can be mediated by drug efflux when Cdr or Mdr transporter, or both, are overexpressed [34,35]. In fact, the switching between a susceptible to a resistant phenotype depends much more on the number of pumps present at the fungal cell membrane [35] rather than to the formation of hyperactive pumps. This addressed the question of the regulation of the efflux transport in Candida. Both genes CDR1 and CDR2 possess in their promoter a cisacting drug-responsive element (DRE) necessary for their upregulation. The DRE box is made up of repeats of 50 CGG-30 triplets that constitute the recognition site of transcriptional activators belonging to the Zn(2)-Cys(6) family. TAC1 was the first transcriptional activator of genes involved in efflux transport to be characterized in C. albicans [4]. It is localized to the nucleus and upregulates both CDR1 and CDR2 by binding the DRE box located in the promoters. The deletion of TAC1 is sufficient to abolish the upregulation of CDR2 but not that of CDR1, suggesting the interplay of other cisacting and/or trans-acting factors for the regulation of CDR1. The existence of several naturally occurring TAC1 alleles in C. albicans was then demonstrated, encoding transcriptional factors more or less efficient to up-regulate pump efflux. These alleles differed from each other by several SNPs. Both the presence of specific mutations gain-of-function (GOF) in TAC alleles and the loss of heterozygosity (LOH) yielded C. albicans strains homozygous for hyperactive TAC1 alleles, that were responsible for high-level resistance to azole antifungals [5]. A comparable work was done to identify Mrr1p, a zinc cluster transcriptional regulator of MDR1 [20]. Inactivation of MRR1 was sufficient to abolish the fluconazole resistance mediated by Mdr1p in clinical C. albicans isolates. For both TAC1 and MRR1, mutations gain-of-function generating hyperactive alleles, and subsequent loss of heterozygosity in C. albicans [6] and C. dubliniensis [26], seem to be the two

T. Noël mechanisms that are under selection pressure to generate azole resistant clinical isolates.

Genome plasticity and gene expression in C. albicans: whole and partial aneuploidy, and isochromosome formation The diploid genome of C. albicans has a total size of 32-Mb organized in 2  8 chromosomes (16 Mb per haploid genome). Candida albicans exhibits a strong genomic plasticity that allows it to play with the quantity of genetic information according to the environmental pressure. Accordingly, chromosome truncations and translocations are frequent, probably mediated by mitotic recombination between major repeat sequence (MRS) in the genome. Another important source of variability comes from aneuploidy, which is the consequence of the gain (2n + 1) or of the loss (2n-1) of a whole chromosome, or, in the case of partial aneuploidy, of a segment of a chromosome [29]. Some of these chromosome rearrangements were observed and characterized in the case of azole drug resistance. Using Comparative Genome Hybridization (CGH) arrays, a global technique that permits the quantification of gene copy number at all loci (and more generally of the quantity of DNA along chromosome arms), a study demonstrated that clinical and laboratory isolates of C. albicans resistant to fluconazole were aneuploid for the chromosome 5 [27]. About one third of the fluconazoleresistant strains were trisomic for the chromosome 5, half of them having a partial aneuploidy of the left arm of the chromosome 5. Partial aneuploidy was achieved by formation of the isochromosome I5(L), an abnormal chromosome made up by two identical left arms flanking the centromere, and formed accidentally after a break-induced recombination event. Among other genes, the left arm of the chromosome 5 bears the genes ERG11, encoding the target of azoles, and TAC1, the transcription regulator of the ABC efflux pumps [27]. Using another experimental approach based on the cartography of genetic markers, trisomy of the chromosome 5 and LOH was also reported in Laboratory strains of C. albicans which developed fluconazole resistance when grown in media supplemented with the drug [5]. The duplication of ERG11 and TAC1 contributes greatly to azole resistance through overexpression of the genes, and confers fitness to the resistant isolates when cultivated under fluconazole selection pressure [28]. However, isochromosomes are very instable and can be rapidly lost, notably in absence of selection pressure, and the strain recovers concomitantly antifungal susceptibility. Thus, the selective advantage conferred by aneuploidy is punctual, and is lost rapidly when C. albicans is in a drug-free environment.

Conclusion The main molecular events at the origin of the development of resistance to azole antifungals are non synonymous mutations of the ERG11 gene, encoding the target protein of azole, and of the transcriptional regulators genes of the zinc cluster family: UPC2, that controls expression of the ERG genes, and TAC1 and MRR1 which are transcriptional activators of drug efflux pumps. Depending on the mutation and

Mechanisms of azole resistance in Candida yeasts on the corresponding amino acid substitution in the protein, the levels of antifungal resistance are variable, and often, a combination of different mutational events are necessary to confer a significant selective advantage to a Candida strain exposed to azoles. Such combinations of distinct molecular events leading to fluconazole resistance were already known as they were described in the past in VIH+ patients suffering from oropharyngeal candidiasis [34]. Mutations affecting either ERG11 or TAC1 have the potential to confer high resistance levels and cross-resistance to all azole antifungals. Loss of heterozygosity is often required after mutation in diploid species, so that all alleles at a given locus are converted to a mutant form. In haploid species, however, mutations are more easily expressed and does not necessarily need additional mechanism to confer a resistant phenotype. A second molecular mechanism can arise from gene duplication and overexpression after aneuploidization. This phenomenon is now well characterized in the diploid species C. albicans, but little is known for the other Candida species, notably the haploid species. However, as gene duplication of ERG11 was already described in C. glabrata [17], it is thus probable that resistance arising from aneuploidization also occurs in haploid species. In spite of the fact that azole resistance in Candida yeasts can develop through very different cellular mechanisms, mutation event is the key underlying molecular mechanism. Fortunately, the frequency of mutation is rare enough for giving us the opportunity to continue to use azole molecules in antifungal therapy.

Disclosure of interest The author declare that he has no conflicts of interest concerning this article.

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