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Molecular mechanisms of fluconazole resistance in Candida dubliniensis isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

ABSTRACT: 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.

SUBMITTER: Perea S

PROVIDER: S-EPMC127221 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Molecular mechanisms of fluconazole resistance in Candida dubliniensis isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

Perea Sofia S López-Ribot José L JL Wickes Brian L BL Kirkpatrick William R WR Dib Olga P OP Bachmann Stefano P SP Keller Suzanne M SM Martinez Marcos M Patterson Thomas F TF

Antimicrobial agents and chemotherapy 20020601 6

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 i ...[more]

PMID: 12019078

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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: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 fluconazole resistance in Candida dubliniensis isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

Publications

Molecular mechanisms of fluconazole resistance in Candida dubliniensis isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

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