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Molecular mechanisms of itraconazole resistance in Candida dubliniensis.

ABSTRACT: It has previously been shown that overexpression of the CdMDR1 gene is a major contributor to resistance in fluconazole-resistant isolates of Candida dubliniensis. However, since CdMdr1p does not mediate transport of other azole drugs such as itraconazole, we investigated the molecular mechanisms of stable resistance to itraconazole obtained in three strains of C. dubliniensis (two with nonfunctional CdCDR1 genes and one with functional CdCDR1 genes) by serial exposure to this antifungal agent in vitro. Seven derivatives that were able to grow on agar medium containing 64 micro g of itraconazole per ml were selected for detailed analysis. These derivatives were resistant to itraconazole, fluconazole, and ketoconazole but were not cross resistant to inhibitors. CdMDR1 expression was unchanged in the seven resistant derivatives and their parental isolates; however, all seven derivatives exhibited increased levels of CdERG11 expression, and six of the seven derivatives exhibited increased levels of CdCDR1 expression compared to the levels of expression by their respective parental isolates. Except for one derivative, the level of rhodamine 6G efflux was decreased in the itraconazole-resistant derivatives compared to the level of efflux in their parental isolates, suggesting altered membrane properties in these derivatives. Analysis of their membrane sterol contents was consistent with a defective sterol C5,6-desaturase enzyme (CdErg3p), which was confirmed by the identification of mutations in the alleles (CdERG3) encoding this enzyme and their lack of functional complementation in a Saccharomyces cerevisiae erg3 mutant. The results of this study show that the loss of function of CdErg3p was the primary mechanism of in vitro-generated itraconazole resistance in six of the seven the C. dubliniensis derivatives. However, the mechanism(s) of itraconazole resistance in the remaining seventh derivative has yet to be determined.

SUBMITTER: Pinjon E

PROVIDER: S-EPMC166077 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Molecular mechanisms of itraconazole resistance in Candida dubliniensis.

Pinjon Emmanuelle E Moran Gary P GP Jackson Colin J CJ Kelly Steven L SL Sanglard Dominique D Coleman David C DC Sullivan Derek J DJ

Antimicrobial agents and chemotherapy 20030801 8

It has previously been shown that overexpression of the CdMDR1 gene is a major contributor to resistance in fluconazole-resistant isolates of Candida dubliniensis. However, since CdMdr1p does not mediate transport of other azole drugs such as itraconazole, we investigated the molecular mechanisms of stable resistance to itraconazole obtained in three strains of C. dubliniensis (two with nonfunctional CdCDR1 genes and one with functional CdCDR1 genes) by serial exposure to this antifungal agent i ...[more]

PMID: 12878500

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Project description:Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

Project description:Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.

Project description:The present study investigated the role of the Candida dubliniensis CdCDR1 and CdCDR2 genes in the development of fluconazole resistance. The C. dubliniensis CdCDR1 gene was 92% identical at the nucleotide sequence level to the corresponding C. albicans gene. However, 58% (14 of 24) of C. dubliniensis genotype 1 isolates tested harbored a nonsense mutation in the CdCDR1 open reading frame that converted codon 756 (TAT) to a TAG translational stop codon. Analysis of five of these C. dubliniensis isolates by Western immunoblotting showed that they expressed a truncated 85-kDa CdCdr1p compared to the full-length 170-kDa CdCdr1p. Expression of CdCDR1 alleles from six C. dubliniensis isolates in a pdr5 Saccharomyces cerevisiae strain revealed that CdCDR1 alleles from three isolates that encoded truncated proteins were unable to confer resistance to drugs and antifungals. However, reassignment of the TAG sequence at codon 756 to TAT (encoding tyrosine) in an allele from strain CD36 conferred the ability to mediate resistance to multiple drugs. Fluconazole-resistant isolates of C. dubliniensis harboring functional alleles of CdCDR1 were found to exhibit two- to ninefold-higher levels of CdCDR1 mRNA than did matched fluconazole-susceptible isolates. By comparison, levels of CdMDR1 expression ranged from approximately 50- to 100-fold greater in resistant isolates. Fluconazole resistance was also identified in isolates harboring nonfunctional CdCDR1 alleles, but resistance in these isolates was only associated with increased CdMDR1 expression. Targeted disruption of two functional alleles of CdCDR1 in a fluconazole-resistant derivative of C. dubliniensis that overexpressed both CdCDR1 and CdMDR1 revealed that although CdCDR1 was important for mediating reduced susceptibility to itraconazole and ketoconazole, there was no affect on fluconazole susceptibility in the double mutant. Evidence presented in this study reveals that CdCDR1 is not essential for the development of fluconazole resistance in C. dubliniensis.

Project description:There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis strains also have an intron that is larger than that in genotype B C. albicans strains but that is in the same location. PCR designed to span this region resulted in a single product for C. albicans genotype A (450 bp), B (840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis (1,080 bp), whereas the C. albicans genotype C isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product identical to that given by C. dubliniensis. These results indicate that those strains previously designated C. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoidea and C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that there is a correlation between the Candida groups and antifungal susceptibility.

Project description:Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [3H] fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible delta pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [3H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.

Dataset Information

Molecular mechanisms of itraconazole resistance in Candida dubliniensis.

Publications

Molecular mechanisms of itraconazole resistance in Candida dubliniensis.

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