The transcription factor RttA contributes to sterol regulation and azole resistance in Aspergillus fumigatus

Major mechanisms of azole resistance in Aspergillus fumigatus involve overexpression of the azole target protein encoded by cyp51A. The elevated expression originates from the duplication of an enhancer element in its promoter, serving as a binding platform for AtrR and SrbA, two central transcription factors that orchestrate the activation of ergosterol biosynthesis genes and azole resistance. Alongside AtrR and SrbA, several other transcription factors were shown to be crucial to mediate azole tolerance. Here, we unveil RttA as a further protein involved in the regulation of ergosterol biosynthesis and azole resistance in A. fumigatus. Because the gene was wrongly annotated, its encoded protein remained a protein with unclear function. Based on mapped RNA-seq reads, the coding sequence was corrected, yielding a DNA-binding protein comprising a Zn₂Cys₆ binuclear zinc cluster. Domain analysis and structural comparisons implied similarity between RttA and Neurospora crassa NcSR and yeast Upc2, both involved in sterol regulation and azole resistance. Through deletion and overexpression of rttA, we confirm its role in azole resistance. Transcriptional profiling of atrR, srbA, and rttA deletion mutants revealed that rttA expression depends on both AtrR and SrbA. In addition, our analyses uncovered a positive regulatory role for RttA in the expression of efflux pump-encoding genes and sterol homeostasis through activation of erg6 expression. In agreement, the lack of rttA led to a substantial accumulation of the Erg6 substrate lanosterol. Collectively, this work elucidates RttA as a transcription factor in the clinically important fungal pathogen A. fumigatus involved in the regulation of ergosterol biosynthesis and azole tolerance.IMPORTANCEAzole antifungals are frontline treatments against Aspergillus fumigatus, a major cause of life-threatening fungal infections. Resistance to azoles is a growing concern, often linked to increased expression of cyp51A, which encodes the azole target enzyme. This upregulation depends on the transcription factors AtrR and SrbA, key activators of ergosterol biosynthesis genes. Here, we identify and characterize a previously misannotated gene, rttA, encoding a Zn₂Cys₆ transcription factor structurally related to Upc2 and NcSR, sterol regulators in yeast and Neurospora crassa. Functional analyses, including gene deletion, overexpression, and transcriptomics, show that RttA promotes azole resistance and regulates sterol homeostasis by activating erg6, encoding sterol C24-methyltransferase. Loss of rttA leads to lanosterol accumulation, indicating disrupted ergosterol biosynthesis. Moreover, rttA expression depends on both AtrR and SrbA, placing RttA within their regulatory network. Our findings offer new insight into sterol regulation and antifungal resistance in A. fumigatus, highlighting RttA as a novel regulator.