The Cytochrome P450 Reductase CprA is a Rate-Limiting Factor for Cyp51A-mediated Azole Resistance in Aspergillus Fumigatus.

Azole antifungals remain the "gold standard" therapy for invasive aspergillosis. The world-wide emergence of isolates resistant to this drug class, however, developed into a steadily increasing threat to human health over the past years. In Aspergillus fumigatus, major mechanisms of resistance involve increased expression of cyp51A encoding one of two isoenzymes targeted by azoles. Yet, the level of resistance caused by cyp51A upregulation, driven by either clinically relevant tandem repeat mutations within its promoter or the use of high expressing heterologous promoters, is limited. Cytochrome P450 enzymes such as Cyp51A rely on redox partners that provide electrons for their activity. A. fumigatus harbors several genes encoding putative candidate proteins including two paralogous cytochrome P450 reductases, CprA and CprB, and the cytochrome b(5) CybE. In this work, we investigated the contribution of each cprA, cprB, and cybE overexpression to cyp51A-mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter PxylP, we conditionally expressed these genes in combination with cyp51A, revealing cprA as the main limiting factor. Similar to this approach, we overexpressed cprA in an azole-resistant background strain carrying a cyp51A allele with TR34 in its promoter, which led to a further increase in its resistance. Employing sterol measurements, we demonstrate an enhanced eburicol turnover during upregulation of either cprA or cyp51A, which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance.