AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus

Abstract

Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1. In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis. IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus. Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.

A 23 bp cyp51A Promoter Deletion Associated With Voriconazole Resistance in Clinical and Environmental Isolates of Neocosmospora keratoplastica

“…Expression and/or upregulation of ATP-binding cassette (ABC) transporters is also frequently associated with azole resistance in fungi (Lamping et al, 2009;Panapruksachat et al, 2016;Watanasrisin et al, 2016;Paul et al, 2019). In A. fumigatus, the AtrR TF binds to, and regulates, both cyp51A and the ABC transporter abcG1.…”

Resistance in the Environmental Pathogenic Fungus <i>Aspergillus fumigatus</i>

“…The repeat is 34-, 46-, or 53-bp long and located approximately 300 bp upstream of the cyp51A start codon. The region contains AtrR-and SrbA-binding elements [6][7][8] , which regulate cyp51A expression. In addition to the duplication of the region upregulating cyp51A expression, amino acid substitutions contribute to azole resistance, except in the TR53-type strain, which contains no substitution in cyp51A.…”

The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

“…We have shown that loss of the NCT complex leads to a large increase in MIC to the azoles, most notably for posaconazole where we observed a shift in MIC from 0.25 mg/L to >16 mg/L. This phenotype is associated with transcriptional dysregulation of the ergosterol biosynthetic pathway directly and indirectly via srbA 10 , atrR 11,15 and hapC 12 , an increase in cellular ergosterol levels (c. 1.6-fold) and an increase in levels of the Cdr1B azole transporter (2.4-fold) (see model in Fig. 9).…”

“…The repeat duplicates the DNA-binding site leading to SrbA-mediated upregulation of cyp51A and a concomitant increase in azole resistance. AtrR, a Zn2-Cys6 transcription factor, also positively regulates sterol biosynthesis and directly binds the cyp51A promoter at the TR site, and additionally the promoter of an azole exporter, cdr1B 11,15 . The CCAAT-binding domain complex CBC, a heterotrimer comprising HapB, HapC and HapE, is a negative regulator of sterol biosynthesis directly binding the promoters of 14 ergosterol biosynthetic genes, including cyp51A 12 .…”