The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter.

We report the cloning and functional analysis of a third member of the CDR gene family in Candida albicans, named CDR3. This gene codes for an ABC (ATP-binding cassette) transporter of 1,501 amino acids highly homologous to Cdr1p and Cdr2p (56 and 55% amino acid sequence identity, respectively), two transporters involved in fluconazole resistance in C. albicans. The predicted structure of Cdr3p is typical of the PDR/CDR family, with two similar halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six predicted transmembrane segments. Northern analysis showed that CDR3 expression is regulated in a cell-type-specific manner, with low levels of CDR3 mRNA in CAI4 yeast and hyphal cells, high levels in WO-1 opaque cells, and undetectable levels in WO-1 white cells. Disruption of both alleles of CDR3 in CAI4 resulted in no obvious changes in cell morphology, growth rate, or susceptibility to fluconazole. Overexpression of Cdr3p in C. albicans did not result in increased cellular resistance to fluconazole, cycloheximide, and 4-nitroquinoline-N-oxide, which are known substrates for different transporters of the PDR/CDR family. These results indicate that despite a high degree of sequence conservation with C. albicans Cdr1p and Cdr2p, Cdr3p does not appear to be involved in drug resistance, at least to the compounds tested which include the clinically relevant antifungal agent fluconazole. Rather, the high level of Cdr3p expression in WO-1 opaque cells suggests an opaque-phase-associated biological function which remains to be identified.     

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.     

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida . The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 μg mL⁻¹); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1 , CDR2 , MDR1 , encoding for efflux pumps, and ERG11 , encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly ( P >0.05), probably acting as a Cdrp blocker.     

A fourth gene from the Candida albicans CDR family of ABC transporters

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I.1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.     

Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.     

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.     

Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.     

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.     

PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.     

Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties

The expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p- and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucose-dependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux.     

Oral candidosis: characterization of a sample of recurrent infections and study of resistance determinants

Recurrent oral candidosis is a common problem in immunocompromised patients, and it is frequently triggered by resistance induced by antifungal treatment. Knowledge of the mechanisms by which the yeast persists in the host could allow the management of this type of infection. This study used electrophoretic karyotyping and restriction fragment length polymorphism based on the use of 27A probe to study 12 pairs of Candida albicans isolates from patients with recurrent candidosis to distinguish new infections from relapses caused by the same strain responsible for the first episode. Subsequently, RT-PCR was used to evaluate expression of CDR1, CDR2 and MDR1 genes, which are involved in C. albicans azole resistance, in the three pairs that consisted of variants of the same strain. Restriction polymorphism resulted in better discrimination than with karyotyping in defining differences between strains. In one case, RT-PCR allowed us to identify deregulation of efflux pump genes as the possible underlying mechanism in recurrent candidosis. The techniques employed resulted effective for the characterization of recurrent oral candidosis. Broader analysis could help to control better these infections and choose adequate therapy.     

Mechanisms of antifungal resistance

The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have been reported for variants of Saccharomyces cerevisiae and of Ustilago maydis. Tolerance to morpholines is seldom found. Intrinsic resistance to griseofulvin is due to the absence of a prolonged energy-dependent transport system for this antibiotic. Resistance to azole antifungal agents is known to be exceptional, although it does now appear to be increasing in importance in some groups of patients infected with e.g. Candida spp., Histoplasma capsulatum or Cryptococcus neoformans. For example, resistance to fluconazole is emerging in C. albicans, the major agent of oro-pharyngeal candidosis in AIDS patients, after long-term suppressive therapy. In the majority of cases, primary and secondary resistance to fluconazole and cross-resistance to other azole antifungal agents seems to originate from decreased intracellular accumulation of the azoles, which may result from reduced uptake or increased efflux of the molecules. In most C. albicans isolates the decreased intracellular levels can be correlated with enhanced azole efflux, a phenomenon linked to an increase in the amounts of mRNA of a C. albicans ABC transporter gene CDR1 and of a gene (BEN(r) or CaMDR) coding for a transporter belonging to the class of major facilitator multidrug efflux transporters. Not only fluconazole, ketoconazole and itraconazole are substrates for CDR1, terbinafine and amorolfine have also been established as substrates, BEN(r) overexpression only accounts for fluconazole resistance. Other sources of resistance: changes in membrane sterols and phospholipids, altered or overproduced target enzyme(s) and compensatory mutations in the Delta5,6-desaturase.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.