Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and Delta srbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Delta erg25A accumulates more C4-methyl sterol intermediates than Delta erg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Delta erg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of Delta srbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.