Complex and critical roles for the AtrR transcription factor in control of cyp51A expression in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34 or 46 bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34/TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element: SRE) and AtrR binding sites (AtrR response element: ATRE) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34 bp repeat of the TR34 promoter (further 5 from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34 or 46 bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34 or 46 bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications. ImportanceAspergillus fumigatus is the most common human filamentous fungal pathogen. Azole drugs are the current therapy of choice for A. fumigatus but the prevalence of azole resistance is increasing. The main genetic alteration causing azole resistance is a change in the cyp51A gene that encodes the target of these drugs. Azole-resistant cyp51A alleles routinely contain duplications in their promoter regions that cause increased gene transcription. Here, we demonstrate that clinical isolates containing a 34 or 46 bp duplication in the cyp51A promoter required the presence of the transcription factor-encoding atrR gene to exhibit elevated azole resistance. Elimination of transcription factor binding sites in the cyp51A gene have differential actions on expression of the resulting mutant allele. These data dissect the molecular inputs to cyp51A transcription and reveal a complicated function of the promoter of this gene that is critical in azole resistance.