Biochemical identification of a nuclear coactivator protein required for AtrR-dependent gene regulation in Aspergillus fumigatus

Abstract

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn2Cys6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of the both the cyp51A gene encoding the azole drug target enzyme as well as an ATP-binding cassette transporter-encoding gene called abcG1 (aka cdr1B). We have used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associate with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to MudPIT mass spectrometry and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (Nuclear Co-Activator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although did not impact induction of cyp51A transcription. We confirmed association of AtrR and NcaA by co-immunoprecipitation from otherwise wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR. ImportanceAspergillus fumigatus is the major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic and infections associated with these azole resistant isolates linked to treatment failure and worse outcomes. Many of these azole resistant mutant strains contain mutant alleles of the cyp51A gene encoding the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole inducible cyp51A transcription but we know little of the regulation of this transcription factor. Using a biochemical approach, we identify a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.