A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme

Abstract

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14alpha-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14alpha-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.

Figure 1

Description of the late sterol biosynthetic pathway in S. cerevisiae wild type (—), erg11 (---), and erg11 erg25 slu (_-^- _-^- _-^-). Shadowed boxes represent the enzymatic steps involved in this investigation. C-4 demethylation depends upon enzymes encoded by ERG25 and as yet two unidentified additional genes.

Figure 2

(A) Growth responses of slu1, erg25 slu1, and erg11 erg25 slu1 grown aerobically on YPD and anaerobically on YPD with ergosterol. The triple mutant (erg11 erg25 slu1) is suppressed on YPD unlike the erg25 slu1 double mutant. (B) The same result with slu2, erg25 slu2, and erg11 erg25 slu2. All plates were grown for 48 h.

Figure 3

Growth responses of wild-type (WA1), slu1, and four erg25 slu1 transformants grown on YPD, YPD plus ergosterol, and YPD plus 1 μM azoles (clotrimazole, ketoconazole, or itraconazole) to indicate that azoles mimic erg11 mutations and rescue the erg25 slu1 double mutant. All growth was aerobic except on ergosterol where growth was anaerobic. All plates were grown for 48 h.

Figure 4

Growth responses of wild-type (WA1), slu2, and four erg25 slu2 transformants grown under the same conditions as in Fig. 3.

Figure 5

Growth responses of wild-type (WA1), slu1, and four erg25 slu1 transformants grown on YPD, YPD plus ergosterol, and YPD plus azoles (1 μM clotrimazole, ketoconazole, or itraconazole) and hemin (26 μg/ml) which reverses the growth stimulatory effects of the azoles. All growth was aerobic except on ergosterol where growth was anaerobic.

Figure 6

Growth responses of wild-type (WA1), slu2, and four erg25 slu2 transformants grown under the same conditions as in Fig. 5.