Azole-resistant Candida Albicans Research Articles

Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis.

While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis.

The Rpd3/Hda1 family of histone deacetylases regulates azole resistance in Candida albicans.

The histone deacetylase (HDAC) has recently been linked to the morphogenesis and virulence of yeast. However, the effects of HDAC on antifungal susceptibility are not well understood. We sought to characterize the action of histone deacetylation on azole resistance in Candida albicans and its possible mechanism of action. A total of 40 C. albicans strains were studied. Azole susceptibility with or without trichostatin A (TSA) was determined according to the CLSI microdilution method. The null mutants of HDA1 and RPD3 (genes targeted by TSA) were also investigated using drop-plate assays and a rapid acquisition of adaptation to the azole test. Transcriptional levels of HDAC genes and efflux genes were quantified using RT-PCR for both the basal and fluconazole-induced conditions. The inhibition of HDACs by TSA (0.25 mg/L) markedly reduced the trailing growth and the growth of most C. albicans strains. Trailing growth for C. albicans strains was decreased from 2-fold to 256-fold at 48 h. The deletion of HDA1 or RPD3 increased the susceptibility to azoles compared with the WT strain. The expression of HDA1 and RPD3 was up-regulated to different levels, and returned to the level of the susceptible parental strain when stable resistance had formed during the course of acquired fluconazole resistance both in vitro and in vivo. Efflux genes were poorly expressed in mutant strains compared with those of the WT strain. Our results indicate the important role of the Rpd3/Hda1 family in the development of azole resistance in C. albicans. Histone deacetylation may govern the expression of genes related to the early stages of adaptation to azole stress, such as efflux pump genes.

Targeting the Adaptability of Heterogeneous Aneuploids

Aneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population with superior adaptability. We show here, using a combination of experimental data and a general stochastic model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an "evolutionary trap" (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition "channeling" a karyotypically divergent population into one with a predominant and predictably drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma.

Contribution of Clinically Derived Mutations in ERG11 to Azole Resistance in Candida albicans

In Candida albicans, the ERG11 gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations in ERG11 that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. Although ERG11 mutations have been observed in clinical isolates, the specific contributions of individual ERG11 mutations to azole resistance in C. albicans have not been widely explored. We sequenced ERG11 in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation in ERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinct ERG11 alleles were recovered, with 10 ERG11 alleles containing a single amino acid substitution. We then characterized 19 distinct ERG11 alleles by introducing them into the wild-type azole-susceptible C. albicans SC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥ 4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations in ERG11 are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.

ABC transporters coupled with the elevated ergosterol contents contribute to the azole resistance and amphotericin B susceptibility

Most screening approaches produce compounds that target survival genes and are likely to generate resistance over time. Simply having more drugs does not address the potential emergence of resistance caused by target mutation, drug efflux pumps over-expression, and so on. There is a great need to explore new strategies to treat fungal infections caused by drug-resistant pathogens. In this study, we found that azole-resistant Candida albicans with CaCDR1 and CaCDR2 over-expression is hypersensitive against amphotericin B (AmB) by our high throughput synergy screening (HTSS). In contrast, Δcdr1 and Δcdr2 knockout strains were resistant to AmB. Moreover, clinical isolates with increased expression of CaCDR1 and CaCDR2 demonstrated susceptibility to AmB, which can also synergize with the efflux pumps inducer fluphenazine (FPZ). Finally, the increased drug susceptibility to AmB in azole-resistant C. albicans with drug efflux pumps over-expression was consistent with the elevated expression of CaERG11 and its associated ergosterols in clinical isolates. Our data implies that the level of ergosterol contents determines the susceptibility to azoles and AmB in C. albicans. Deep understanding of the above mechanisms would offer new hope to treat drug-resistant C. albicans.

Berberine Inhibits Fluphenazine-Induced Up-Regulation of <i>CDR1</i> in <i>Candida albicans</i>

Over-expression of the Candida drug resistance gene CDR1 is a common mechanism generating azole-resistant Candida albicans in clinical isolates. CDR1 is transcriptionally activated through the binding of the transcription factor Tac1p to the cis-acting drug-responsive element (DRE) in its promoter. We previously demonstrated that the combination of fluconazole (FLC) and berberine (BBR) produced significant synergy when used against FLC-resistant C. albicans in vitro. In this study, we found that BBR inhibited both the up-regulation of CDR1 mRNA and the transport function of Cdr1p induced by fluphenazine (FNZ). Further, electrophoretic mobility shift assays suggested that the transcription activation complex of protein-DRE was disrupted by BBR, and electrospray ionization mass spectrometry analysis showed that BBR bound to the DRE of CDR1. Thus we propose that BBR inhibits the FNZ-induced transcriptional activation of CDR1 in C. albicans by blocking transcription factor binding to the DRE of CDR1. These results contribute to our understanding of the mechanism of synergistic effect of BBR and FLC.

Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans

Several and often combined mechanisms can lead to acquired azole resistance in Candida albicans and subsequent therapeutic failure. The aim of this study was to provide a complete overview of the molecular basis of azole resistance in a set of six C. albicans clinical isolates recovered from patients who failed azole therapy. For this purpose, expression levels of CDR1, MDR1 and ERG11 were investigated by reverse transcription PCR (RT-PCR) together with amplification and sequencing of the genes encoding their transcription factors TAC1, MRR1 and UPC2. In all, the data underline that azole resistance in this set of clinical isolates results from distinct, often combined, mechanisms, being mostly driven by CDR1 and/or MDR1 active efflux. We show that gain-of-function (GOF) mutations in the transcription-factor-encoding genes TAC1, MRR1 and UPC2 are a common event in azole-resistant C. albicans clinical isolates. In addition, together with the finding that these genes are highly permissive to nucleotide changes, we describe several novel mutations that could act as putative GOF mutations involved in fluconazole resistance.

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole‐resistant Candida albicans strains

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm⁻²). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log₁₀ CFU reduction (0.5 McFarland) was 0.62 μmol l⁻¹ for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l⁻¹. Increasing the fluence to 37 J cm⁻² allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log₁₀ reduction (4 McFarland). H₂O₂ was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas ¹O₂ was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

Pyridone Alkaloids from a Marine-Derived Fungus, Stagonosporopsis cucurbitacearum, and Their Activities against Azole-Resistant Candida albicans

Four new 4-hydroxy-2-pyridone alkaloids, didymellamides A-D (1-4), were isolated from the marine-derived fungus Stagonosporopsis cucurbitacearum. The structures of 1-4 were elucidated from spectroscopic data (NMR, MS, and IR), and the absolute configuration of 1 was determined by X-ray diffraction analysis. Didymellamide A (1) exhibited antifungal activity against azole-resistant Candida albicans.

In vitro Synergistic Effects of Anthracycline Antitumor Agents and Fluconazole Against Azole-Resistant Candida albicans Clinical Isolates

The number of azole-resistant Candida albicans clinical isolates is increasing. This study searched for compounds that are functionally synergistic with fluconazole against azole-resistant C. albicans strains. Synergistic effects were evaluated using the checkerboard method in a time-kill study using anthracycline antitumor agents and azole-resistant C. albicans strains. Of the five anthracycline agents examined, aclarubicin by itself had antifungal effects, whereas daunorubicin, doxorubicin, epirubicin, and idarubicin did not show antifungal effects alone, but did exert dose- and timedependent synergistic effects with fluconazole against the C. albicans strains. No antitumor agent other than anthracycline exhibited an anti-Candida effect. Anthracycline compounds may therefore be useful as seeds for development of new antifungal agents.

The effect of histatin 5, adsorbed on PMMA and hydroxyapatite, on Candida albicans colonization

The limited number of treatments for oral candidiasis resulted in the emergence of azole-resistant Candida albicans strains, thus enforcing the need for novel antifungal treatments. Although histatin 5 (H5) demonstrates antifungal activity, its inhibitory effect when adhered to hydroxyapatite and Polymetylmethacrylate (PMMA) surfaces, resembling conditions of the in vivo pellicle, remains unexplored. The objective of this in vitro study was to determine whether surface-adhered H5 inhibits the colonization of C. albicans on hydroxyapatite and/or PMMA. The C. albicans assay involved developing a mono-protein pellicle (either H5 or albumin) on hydroxyapatite and PMMA discs, introducing C. albicans and counting the number of adhered cells, throughout time, using scanning electron microscopy. A negative binomial statistical model and the Tukey-Kramer test were used for statistical analysis, with p < 0.01 indicating significance. H5-coated PMMA had significantly reduced number of cells compared to albumin-coated PMMA at 30, 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly in 90 and 1440 min (p < 0.0001). Similarly, H5-coated hydroxyapatite had significantly fewer cells compared to the albumin-coated surface at 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly at 30, 90 and 1440 min (p < 0.0001). In conclusion, C. albicans colonization was most inhibited by PMMA and hydroxyapatite-adhered H5 after 1440 min, illustrating the time-dependent effect of H5. In addition, yeast cells colonized albumin-coated PMMA, while dense hyphal networks formed on albumin-coated hydroxyapatite, suggesting that C. albicans morphology is influenced by the surface available for albumin adhesion.

A case series of the management of symptomatic azole-resistant candida

Patients with symptomatic azole-resistant Candida albicans or non-albicans candida are difficult to manage. Treatment is largely anecdotal due to the relatively small number of patients. We present six case reports which highlight our own observation in clinical practice including four patients who were treated successfully with topical amphotericin B/flucytosine vaginal gel for 14 days (Stoke-on-Trent formula).

Mutation analysis of ERG 11 gene in Candida albicans isolates from patients with acquired immunodeficiency syndrome (AIDS)

Objective To investigate the relationship between the mutation of ERG11 gene,a target of azole antifungal drugs (fluconazole,itraconazole,voriconazole),and azole-resistance in Candida albicans isolates from patients with AIDS.Methods Ninety-three Candida albicans strains were isolated from patients with AIDS.DNA was extracted from these isolates,and ERG11 gene was amplified by PCR followed by bidirectional sequencing.DNAman software was used to compare the resultant sequence with the reference sequence of ERG11 gene (GenBank accession no.X13296).Then,different base sequences were translated into amino acid sequences to determine whether missense mutations occured.Results A total of 40 mutation sites were identified in these isolates,including 27 silent mutations and 13 missense mutations.One or no missense mutation was detected in Candida albicans strains resistant to 1 antifungal agent,while those resistant to 2 or 3 antifungal agents simultaneously harbored 2 or 3 missense mutations.Conclusion The missense mutations in ERG11 gene are probably connected with azole resistance in Candida albicans. Key words: Candida albicans; Genes, ERG11; Fluconazole; Itraconazole; Voriconazole

Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

CaIPF14030 negatively modulates intracellular ATP levels during the development of azole resistance in Candida albicans.

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. We investigated the role of CaIPF14030 during the development of azole resistance in C albicans. The expression of CaIPF14030 was measured by quantitative RT-PCR, and CaIPF14030 was disrupted by the hisG-URA3-hisG (URA-blaster) method. The sensitivity of C albicans to azoles was examined using a spot assay, and the intracellular ATP concentrations were measured by a luminometer. CaIPF14030 expression in C albicans was up-regulated by Ca(2+) in a calcineurin-dependent manner, and the protein was overexpressed during the stepwise acquisition of azole resistance. However, disruption or ectopic overexpression of CaIPF14030 did not affect the sensitivity of C albicans to azoles. Finally, we demonstrated that disruption of CaIPF14030 significantly increased intracellular ATP levels, and overexpression significantly decreased intracellular ATP levels in C albicans. CaIPF14030 may negatively modulate intracellular ATP levels during the development of azole resistance in C albicans.

The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans

To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole-resistant (azole-R) clinical isolates of Candida albicans. A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole-R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC-resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by Delta E models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time-killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Synergistic activity of HNK and FLC against clinical isolates of FLC-resistant C. albicans was observed in vitro and in vivo. This report might provide a potential therapeutic method to overcome the problem of drug-resistance in C. albicans.

Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

BackgroundAmino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.ResultsThe RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.ConclusionThe sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.

Proteomic analysis of Mrr1p‐ and Tac1p‐associated differential protein expression in azole‐resistant clinical isolates of Candida albicans

Azole resistance in Candida albicans is frequently caused by the overexpression of multi-drug efflux pump genes MDR1, CDR1, and CDR2 due to gain-of-function mutations in the zinc cluster transcription factors Mrr1p and Tac1p. In this study, we performed a comparative proteomic analysis to identify proteins whose expression level is influenced by these transcription factors. Both 2-DE and PMF were used to examine the expression profiles of six pairs of matched C. albicans isolates carrying gain-of-function mutations in either MRR1 or TAC1 resulting in the overexpression of either MDR1 or CDR1 and CDR2. Using this approach, 17 differentially expressed proteins were identified in the MDR1-overexpressing isolates, while 14 were identified in the isolates that overexpress CDR1 and CDR2. Furthermore, we found that the expression of many of these proteins was increased in a wild-type strain of C. albicans after the introduction of a gain-of-function allele of MRR1 or TAC1. Moreover, disruption of MRR1 and TAC1 in isolates carrying gain-of-function mutations resulted in decreased expression of these proteins, confirming their regulation by Mrr1p or Tac1p. Several proteins involved in heat shock and carbohydrate metabolism were differentially expressed in all clinical isolate sets, but these proteins were not dependent upon either Tac1p or Mrr1p.

RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans.

The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl(2)- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca(2+) chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl(2). Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans.