Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is one of the deadliest fungal species causing hundreds of thousands of deaths each year. As azoles provide the preferred first-line option for treatment of Aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for those infected with a resistant isolate constitutes a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene which encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding-complex (CBC) and HapX that cooperate in the regulation of genes involved in iron-consuming pathways including cyp51A. Here, we studied the regulatory contribution of SrbA, AtrR, CBC and HapX binding sites on cyp51A expression and azole resistance during different iron availability employing promoter mutational analysis and protein/DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further uncover the HapX binding site as repressor element the disruption of which elevates cyp51A expression and azole resistance regardless of iron availability.

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

“…Our previous analyses of both the abcG1 and cyp51A promoters may help explain the differential effects of the ncaAΔ allele on these two genes. Transcription of abcG1 is highly dependent on AtrR activity while expression of cyp51A involves both AtrR as well as the key sterol regulatory transcription factor SrbA (10, 11, 14, 28, 29). NcaA may not be involved in AtrR-dependent activation at the cyp51A promoter or the partially compromised phenotype triggered by the ncaAΔ allele may be suppressed by the presence of normal SrbA.…”

Transcription factor FfmA interacts both physically and genetically with AtrR to properly regulate gene expression in the fungusAspergillus fumigatus

“…The TR34 repeat in the cyp51A promoter has been found to cause an increased level of gene transcription of this gene (MELLADO et al 2007). The level of cyp51A transcription is a key determinant of azole resistance and is determined in part by the cooperative function of the transcriptional activator proteins SrbA and AtrR (KUHBACHER et al 2022;PAUL et al 2022c). SrbA was discovered on the basis of its similarity to the sterol response element-binding protein from mammalian cells and is a key regulator of ergosterol biosynthetic gene expression (WILLGER et al 2008).…”