The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans

Abstract

Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the human immunodeficiency virus (HIV)-infected population. The target enzyme of the azole drugs is lanosterol 14alpha demethylase (Erg16p), a cytochrome P-450 enzyme in the biosynthetic pathway of ergosterol. Biochemical analysis demonstrates that Erg16p became less susceptible to fluconazole in isolate 13 in a series of isolates from an HIV-infected patient. PCR-single-strand conformation polymorphism (PCR-SSCP) analysis was used to scan for genomic alterations of ERG16 in the isolates that would cause this change in the enzyme in isolate 13. Alterations near the 3' end of the gene that were identified by PCR-SSCP were confirmed by DNA sequencing. A single amino acid substitution (R467K) that occurred in isolate 13 was identified in both alleles of ERG16. Allelic differences within the ERG16 gene, in the ERG16 promoter, and in the downstream THR1 gene were eliminated in isolate 13. The loss of allelic variation in this region of the genome is most likely the result of mitotic recombination or gene conversion. The R467K mutation and loss of allelic variation that occur in isolate 13 are likely responsible for the azole-resistant enzyme activity seen in this and subsequent isolates. The description of R467K represents the first point mutation to be identified within ERG16 of a clinical isolate of C. albicans that alters the fluconazole sensitivity of the enzyme.