Fig. 2: Erg251 serves as the major sterol C4-methyl oxidase under EHC in divergent fungal pathogens.

a Schematic of ergosterol biosynthesis. b ERG251 alleles (A or B) were expressed from the native promoter. Gene re-introduction and promoter replacement were homozygous unless otherwise specified. c Spotting assay showing O₂-level contingent growth of erg251 on SD. d Sterol profiles of strains grown on SD agar ± 20 µg mL⁻¹ DOX analyzed by GC-MS after 8-h AC or 24-h EHC incubation. ns not significant (p-values: 0.88; 0.80; 0.42) e The tetO-promoter was used to drive ERG25 in WT (ERG251) and erg251 backgrounds. AC growth was assessed on YPD ± 20 µg mL⁻¹ DOX. f Sterol profiles of the erg251 tetO-ERG25 strain after 24-h EHC growth on SD agar ± 20 µg mL⁻¹ DOX. g Spotting assay showing ERG25 is dispensable for fitness. h Expression of ERG251 was assessed after 6-h of AC or EHC growth on YPD. i Immunoblotting comparing expression of Erg251-GFP and Erg25-GFP from their native promoter after 9-h EHC growth on YPD. Loading of the Erg251-GFP sample was titrated. *: adjusted image for visualizing Erg25-GFP signal; Coomassie Blue Staining (CBS). j Sequence and position of putative Upc2 binding sites. Highlighted “CG” were changed to “AT” in the mutant promoter (pupc2_mut.) and expression measured after 6-h EHC growth on YPD. Analyses in this panel were based on ERG251(B). k–m ERG25(-GFP) expression driven by the ERG251 promoter (p251) or tetO promoter was compared to ERG251(-GFP) using RT-qPCR (k) and immunoblot (l). Growth was tested under different O₂ levels on YPD via spotting assay (m). P-values comparing ERG25 expression are reported in (k). n The erg251-deletion strains re-introduced with p251-driven ERG251 or ERG25 were grown for 24 h on SD agar under EHC. Sterol profiles were analyzed by GC-MS. o Phylogenetic tree of fungal sterol C4-methyl oxidases. p Sterol C4-methyl oxidase genes were expressed from the native C. albicans ERG251 promotor (p251) in an erg251 tetO-ERG25 background (“Parent”). Cell growth was assessed on YPD + 20 µg mL⁻¹ DOX. q Independent C. auris transformants (#1, #2) were tested to assess growth via spotting assays. r Transcript levels were measured after 7-h AC or 9-h EHC growth on YPD. s Graphic summary. d (AC part), f, h, j, k, and r show mean ± SD of biological triplicates examined by unpaired t-tests (two-tailed p-values reported); d (EHC part) and n show biological quadruplicates (mean ± SD) examined by Mann–Whitney tests (two-tailed p-values reported).