Genetic analysis of azole resistance in the Darlington strain of Candida albicans

High-level azole resistance in the Darlington strain of Candida albicans wa s investigated by gene replacement in C. albicans and expression in Sacchar omyces cerevisiae, We sequenced the ERG11 gene, which encodes the sterol C( 14)alpha -demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleuc ine substitution (I471T) not previously reported in the C, albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when ove rexpressed alone and increased azole resistance when added to the Y132H ami no acid sequence in an S. cerevisiae expression system, Replacement of one copy of ERG11 in an azole-susceptible strain of C, albicans with a single c opy of the Darlington ERG11 resulted in expression of the integrated copy a nd a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.