Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles.
Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.
Chau
Andrew S
AS
Schering-Plough Research Institute, 2015 Galloping Hill Rd., K15.4-4700, Kenilworth, NJ 07033, USA.
Mendrick
Cara A
CA
Sabatelli
Frank J
FJ
Loebenberg
David
D
McNicholas
Paul M
PM
D000199:Actins / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001393:Azoles / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics*; D002180:Candidiasis, Oral / Q000188:drug therapy / Q000382:microbiology; D003001:Cloning, Molecular; D003199:Computer Systems; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D009960:Oropharynx / Q000382:microbiology; D006180:Proton-Translocating ATPases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013045:Species Specificity; D014158:Transcription, Genetic; D014230:Triazoles / Q000494:pharmacology / Q000627:therapeutic use
C404682:ACT1 protein, Candida albicans; D000199:Actins; D000935:Antifungal Agents; D001393:Azoles; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014230:Triazoles; C101425:posaconazole; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases