SrbA-regulation of ergosterol biosynthesis in Aspergillus fumigatus : gateway to azole resistance & hypoxia adaptation

Abstract

Aspergillus fumigatus is a human fungal pathogen and the primary cause of Invasive Aspergillosis (IA). A rise in susceptible patient populations has dramatically increased the incidence of IA, and led to the emergence of triazole antifungal drug resistance. Triazoles target Erg11, an enzyme involved in ergosterol biosynthesis. Ergosterol biosynthesis has been widely targeted for antifungal drug development, but little is known about this pathway in A. fumigatus. We have identified a transcription factor, SrbA, which mediates triazole susceptibility, growth in hypoxia and low iron, and virulence during IA. Transcriptional studies identify ergosterol biosynthesis as one of the major genetic targets of SrbA, including erg11 and erg25. In this study, we examined the mechanism of Delta srbA triazole susceptibility. Construction of an erg11A conditional expression strain in the Delta srbA background restored erg11A transcript levels and, consequently, wild-type sensitivity to fluconazole and voriconazole. However, pniiAerg11A-Delta srbA did not restore hypoxia growth or the total ergosterol defect of Delta srbA. Increased accumulation of C4-methyl sterols indicates that the Erg25-step of ergosterol biosynthesis is defective in these strains. A. fumigatus encodes for two C4-demethylases, erg25A and erg25B. Erg25A serves in a primary role over Erg25B, as Delta erg25A accumulates more C4-methyl sterol intermediates than Delta erg25B. That both erg25 genes retain function, and are not limited to a singular substrate is unique in the eukaryotic kingdom. Genetic deletion of both erg25 genes is lethal, and single deletion of these genes revealed alterations in ergosterol biosynthesis. Delta erg25A displayed moderate sensitivity to hypoxia, reactive oxygen species (ROS), and dithiothreitol, but was not required for virulence in a murine model of IA. Erg25 assists in the ability of A. fumigatus to grow in hypoxia, as construction of a strain that constitutively expresses erg25A in the Delta srbA background restored the hypoxia growth defect of Delta srbA. This restoration revealed substantial insufficiencies in pflavA-erg25A-Delta srbA when adapting to hypoxia, as this strain was hypersensitive to cell wall perturbation and ROS. Additionally, restoration of erg25A impacted triazole antifungal susceptibility of Delta srbA, demonstrating a complex feedback system involved in ergosterol biosynthesis. These results demonstrate SrbA's involvement in a dynamic stress adaptation program mediated in part through ergosterol biosynthesis.