Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae

Abstract

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. 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.IMPORTANCE A common target of antifungal drug treatment is the fungal ergosterol biosynthesis pathway. This report helps to identify ergosterol biosynthesis enzymes that have not previously been appreciated as drug targets. The effects of overexpression of each of the 25 ERG genes in S. cerevisiae were analyzed in the presence of six stress agents that target essential cellular processes (cell wall biosynthesis, protein translation, respiration, osmotic/ionic stress, and iron and calcium homeostasis), as well as six antifungal inhibitors that target ergosterol biosynthesis. The importance of identifying cell perturbations caused by gene overexpression or deletion is emphasized by the prevalence of gene expression alterations in many pathogenic and drug-resistant clinical isolates. Genes whose altered expression causes the most extensive phenotypic alterations in the presence of stressors or inhibitors have the potential to be drug targets.

Keywords: Saccharomyces cerevisiae; antifungal drug resistance; ergosterol biosynthesis; ergosterol gene overexpression; ergosterol regulation; stress agents.

FIG 1

Ergosterol biosynthetic pathway. The box on the left diagrams the mevalonate pathway, which can channel products into different biosynthetic pathways. The box in the middle represents the late ergosterol pathway terminating in ergosterol. The box on the right represents an alternate pathway leading to the toxic fungistatic sterol [14α methylergosta 8-24-28 dienol]. Boxed gene names denote nonessential genes. Red names represent antifungal drugs and their targets of inhibition.

FIG 2

Doubling times of strains overexpressing ERG genes. Strains carrying plasmids containing ERG genes were grown in Gal media to induce gene overexpression (A) in the W303-1a strain background and (B) in the BY4741 strain background. Significant values are plotted as gray bars. The genes are listed in their order along the ergosterol biosynthesis pathway.

FIG 3

Iron requirement of strains overexpressing ERG genes. The doubling time of strains overexpressing each of the ERG genes was measured in Gal media alone, in Gal media plus 1 mM ferrozine (black bars), and in Gal media plus 1 mM ferrozine plus 300 µM FeSO₄ (gray bars). The genes are listed in their order along the ergosterol biosynthesis pathway.

FIG 4

Calcium requirement of strains overexpressing ERG genes. The doubling time of strains overexpressing each of the ERG genes was measured in Gal media alone, in Gal media plus 4 mM EGTA (black bars), and in Gal media plus 4 mM EGTA plus 20 mM CaCl₂ (gray bars). The genes are listed in their order along the ergosterol biosynthesis pathway.

FIG 5

Cellular response to osmotic/ionic stress. The doubling time of strains overexpressing each of the ERG genes was measured in Gal media alone or in Gal media with 1.2 M NaCl. The genes are listed in their order along the ergosterol biosynthesis pathway.

FIG 6

Utilization of a nonfermentable carbon source to elucidate respiratory deficiencies. Slow growers (A) and normal growers (B) were grown in Glu media for 24 h and then plated on Glu media (Glu-Glu) or Gly media (Glu-Gly). Strains were also grown in Gal media for 48 h and then plated on Glu media (Gal-Glu) or Gly media (Gal-Gly). The black outlines highlight the strains with a growth deficiency on glycerol media. Blue outlines highlight growth improvement on glycerol media.

FIG 7

Response to cell wall stress. Slow growers (A) and normal growers (B) were grown in Glu media and plated on Glu media (Glu-Glu) in the absence (−CR) or presence (+CR) of 64 µg/ml of Congo red. The strains were also grown in Gal media and plated on Gal media (Gal-Gal) in the absence (−CR) or presence (+CR) of 64 µg/ml of Congo red. The black outlines highlight strains sensitive to CR.

FIG 8

Summary of stress agent phenotypes. The strains whose designations appear in squares demonstrated a phenotype that was altered from the WT phenotype under that condition. "E" signifies an ERG gene, "I1" signifies the IDI1 gene, "H" signifies the HMG genes, and "N1" signifies the NCP1 gene.

FIG 9

Summary of antifungal drug susceptibilities. The strains whose designations appear in squares demonstrated drug susceptibility that was altered from that seen with the WT strain for that drug. The letter E signifies ERG genes, the letter H signifies the HMG genes, and "N1" signifies the NCP1 gene. Δ = gene deletion. Drug abbreviations are indicated with purple text. Green text represents increased susceptibility. Red text represents increased resistance. The presence of identical genes is indicated with boxes with thick borders. The different drugs are indicated with boxes with thin borders.