Contributions of both ATP-binding cassette transporter and Cyp51A proteins are essential for azole resistance in Aspergillus fumigatus

Abstract

While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus. Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.