Abstract
ABSTRACTErgosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme in Saccharomyces cerevisiae were analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.
Highlights
Ergosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway
Formed by association of sterols and sphingolipids and are enriched with many biologically important molecules such as efflux pumps, sodium and potassium pumps, receptors, and nutrient transporters [1, 2]. These microdomains are central to a variety of cellular processes, stress responses, and adaptations to the environment; maintaining lipid rafts is critical for proper functioning of the cells [1]
These enzymes are regulated in part by the zinc-cysteine finger transcription factor paralogs Upc2p/Ecm22p in Saccharomyces cerevisiae and by Upc2p in the pathogenic fungus Candida albicans [3, 4]
Summary
Ergosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. Overexpression or Deletion of ERG Biosynthesis Genes conditions such as by treatment with azoles (Fig. 1). The doubling time of strain W303-1A expressing each of the 25 plasmid-borne ERG genes was analyzed as the strains grew in either noninducing glucose-containing media (Glu media) or Gal media for 96 h.
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