Loss of a conserved C-terminal region of theAspergillus fumigatusAtrR transcriptional regulator leads to a gene-specific defect in target gene expression

Abstract

AbstractTreatment of fungal infections associated with the filamentous fungusAspergillus fumigatusis becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by thecyp51Agene. A common route of azole resistance involves an increase in transcription ofcyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1Bhere calledabcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain inA. fumigatusis required for expression ofabcG1but dispensable forcyp51Atranscription. This domain is also found in several other fungal pathogen AtrR homologues consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA-seq, we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.ImportanceAspergillus fumigatusis the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated withA. fumigatusare the azole compounds. These drugs are well-tolerated and effective but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by thecyp51Agene. A key regulator ofcyp51Agene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologues from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.