Clinically-derived mutations in ERG11 caused decreased fluconazole susceptibility when expressed in a susceptible strain of C. albicans

Purpose: ERG11, which encodes the ergosterol biosynthesis enzyme lanosterol demethylase, can contribute to fluconazole resistance by its overexpression due to GOF mutations in transcription factors like Upc2, or by ERG11 mutations that interfere with binding of azole antifungals.   Although mutations in ERG11 are associated with azole resistance, the specific contribution of these mutations to fluconazole resistance in a C. albicans background has not been explored.  

Methods: In 29 clinical isolates with decreased fluconazole susceptibility, both alleles of ERG11 and UPC2 were sequenced.  We selected 6 mutant alleles (K143R, G464S, Y132F, V488I, G488S and S405F) to express homozygously or with a wild-type allele in an azole-susceptible background strain, SC5314. For the constructed strains, we tested resulting FCZ susceptibility and ERG11 expression by q-RT-PCR.

Results:  We found that 23 of the 29 clinical isolates carried at least one mutation in ERG11 that resulted in an amino acid substitution in the predicted protein sequence. Mutations in ERG11 were observed in isolates that also carried a mutation in UPC2.  Decreased FCZ susceptibility was observed for strains expressing alleles containing the K143R, G464S, Y132F, V488I and S405F substitutions.  When expressed homozygously, the K134R substitution resulted in a 4-fold increase in FCZ MIC as compared to SC5314.  The Y132F substitution increased FCZ MIC by 2-fold.  Likewise, the G464S substitution, the V488I substitution and the S405F substitution each increased FCZ MIC by 1-fold.  Interestingly, distinct mutations in ERG11 resulted in altered ERG11 expression in these constructed strains.  

Conclusion: These findings indicate that mutations in ERG11 are prevalent in a large group of clinical isolates and most ERG11 mutations characterized in our study contribute to decreased fluconazole susceptibility and influence ERG11 expression.  Our sequence data show that ERG11 mutations occur with UPC2 mutations.  We suspect that the collective result of these mutations has a significant impact on fluconazole susceptibility.