The Candida dubliniensis CdCDR1 gene is not essential for fluconazole resistance.
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.
Moran
Gary
G
Microbiology Research Unit, Department of Oral Surgery, Oral Medicine and Pathology, School of Dental Science, Trinity College, University of Dublin, Republic of Ireland.
Sullivan
Derek
D
Morschhäuser
Joachim
J
Coleman
David
D
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics; D000483:Alleles; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D015139:Blotting, Southern; D015153:Blotting, Western; D002175:Candida / Q000187:drug effects* / Q000235:genetics*; D017931:DNA Primers / Q000494:pharmacology; D004271:DNA, Fungal / Q000187:drug effects / Q000235:genetics; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D018390:Gene Targeting; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D015725:Fluconazole