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High mortality rates from invasive aspergillosis in immunocompromised patients are prompting research toward improved antifungal therapy and better understanding of fungal physiology. Herein we show that Aspergillus fumigatus, the major pathogen in aspergillosis, imports exogenous cholesterol under aerobic conditions and thus compromises the antifungal potency of sterol biosynthesis inhibitors. Adding serum to RPMI medium led to enhanced growth of A. fumigatus and extensive import of cholesterol, most of which was stored as ester. Growth enhancement and sterol import also occurred when the medium was supplemented with purified cholesterol instead of serum. Cells cultured in RPMI medium with the sterol biosynthesis inhibitors itraconazole or voriconazole showed retarded growth, a dose-dependent decrease in ergosterol levels, and accumulation of aberrant sterol intermediates. Adding serum or cholesterol to the medium partially rescued the cells from the drug-induced growth inhibition. We conclude that cholesterol import attenuates the potency of sterol biosynthesis inhibitors, perhaps in part by providing a substitute for membrane ergosterol. Our findings establish significant differences in sterol homeostasis between filamentous fungi and yeast. These differences indicate the potential value of screening aspergillosis antifungal agents in serum or other cholesterol-containing medium. Our results also suggest an explanation for the antagonism between itraconazole and amphotericin B, the potential use of Aspergillus as a model for sterol trafficking, and new insights for antifungal drug development.

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.