Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450

Abstract

The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another fluconazole-resistant isolate were homozygotes with respect to CYP51, although the typical wild-type, fluconazole-susceptible C. albicans was a CYP51 heterozygote. These findings suggest that part of the fluconazole-resistant phenotype of C. albicans DUMC136 was acquired through a mutation-prone area of CYP51, an area which might promote the formation of fluconazole-resistant CYP51, along with a mechanism(s) which allows the formation of a homozygote of this altered CYP51 in this diploid pathogenic yeast.

FIG. 1

Biosynthetic pathway for ergosterol in C. albicans. Note that two alternative pathways for conversion of lanosterol to fecosterol exist. Abbreviations: 4,4-DZ, 4,4-dimethylzymosterol; 24-Methylene DHL, 24-methylene-24,25-dihydrolanosterol; 4,4-DF, 4,4-dimethylfecosterol.

FIG. 2

The nucleotide (and deduced amino acid) sequence of the highly substituted region observed in CYP51 clones from wild-type C. albicans ATCC 90028 and fluconazole-resistant C. albicans isolates DUMC136 and S78941. The nucleotides and corresponding amino acids of the ATCC 90028-1 allele which are identical to those of the reported C. albicans CYP51 sequences (17) are fully indicated, and only substituted nucleotides and amino acids are shown for ATCC 90028, DUMC136, and S78941. The underlined region is one of the putative substrate recognition sites of CYP51, named SRS-1 (2). Dashes indicate amino acids identical to those of the ATCC 90028-1, and dots indicate nucleotides identical to those of the ATCC 90028-1 gene.

FIG. 3

RFLPs observed on the 673-bp PCR products from the CYP51 genes of ATCC 90028 and DUMC136. (A) The restriction maps of the 673-bp PCR products (nucleotide positions −15 to 658) from the alleles encoding ATCC 90028-1 and ATCC 90028-2 and from the DUMC136 CYP51 gene. (B) DNA fragments obtained by digestion of the 673-bp PCR products with AfaI (a) or RcaI (b) are shown. The templates used for the PCR amplifications were as follows: lane 1, the cloned ATCC 90028-1 DNA; lane 2, the cloned ATCC 90028-2 DNA; lane 3, whole genomic DNA of C. albicans ATCC 90028; lane 4 to 6, whole genomic DNA of three independent colonies of C. albicans DUMC136. Lane M is the DNA size marker (BglII- and HinfI-digested pBR328).