Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors

Abstract

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.

FIG. 1.

Growth of A. fumigatus cultured in 250 ml of RPMI 1640 medium under aerobic conditions. (A) Time course study showing growth enhancement by human serum. Each data point represents a single determination. (B) Inverse correlation between growth and dissolved oxygen. Data were obtained from 24-h cultures containing various amounts of serum, cholesterol, or drugs. (C) Growth in medium containing 0.4% ethanol (EtOH) with or without ITC. Data represent the means ± standard deviations of three independent determinations. No significant difference (P > 0.05) was found between 0 and 0.4% ethanol-treated cells at the same concentration of ITC.

FIG. 2.

Partial NMR spectra showing peaks used for quantitation. (A) Folch extract of cells cultured in 0.3% serum, showing the H-18 signals for cholesterol, ergosterol, and the internal standard epicoprostanol. (B) Folch extract of a control sample. Strong resolution enhancement was used to separate the peaks of free and esterified ergosterol. (C) The H-19 peaks of free and esterified cholesterol of a Folch extract of cells cultured in 3% serum. (D) The C-19 peaks of free and esterified cholesterol in the ¹³C NMR spectrum (125 MHz) of the same sample as that shown in panel C. (E) The H-18 signals of NSL of cells cultured in 54 μg of cholesterol/ml.

FIG. 3.

¹H NMR spectra showing profiles of the major aberrant sterol intermediates that accumulate upon treatment with ITC and VRC. Samples are from the NSL of the cell pellets. (A) Control culture with no drug treatment. Lanosterol was also below the 1% detection limit in drug-treated cultures. (B) Cells treated with 0.01 μg of ITC/ml. Cells treated with 0.12 μg of VRC/ml (C) or with 0.20 μg/ml VRC (D). Signals at δ 0.701, 0.706, and 0.743 represent unidentified sterols, such as 14α-methylfecosterol.