The Influence of Ergosterol Biosynthesis Inhibition on the Mycelial Growth in Aspergillus flavus

E. Mizusawa, K. Hashimoto, H. Maki, A. Naito

Author address: 

Shionogi & Co., Ltd., Toyonaka, Japan

Abstract: 

Background: Invasive aspergillosis (IA) contributes to significant mortality in immunosuppressed patients. Azoles, known as Cyp51 inhibitors, are used to treat IA. The majority of IA is caused by Aspergillus fumigatus, followed by Aspergillus flavus. The effect of these inhibitors on ergosterol biosynthesis pathway has been studied mostly in A. fumigatus, whereas little has been reported in A. flavus. In this study, we compared the influence of Cyp51 inhibition by voriconazole (VRCZ) on the mycelial growth in A. fumigatus and A. flavus. Methods: A. fumigatus and A. flavus were grown by shaking overnight in YNB broth at 35°C, then VRCZ and 2- 13C-acetate were added to the cultures. After incubation at 35°C for 16 hr, mycelia pellets were harvested by vacuum filtration. Sterols were extracted by n-heptane from the cell pellets. From them, ergosterol, eburicol and abnormal C14-methylated sterols such as obtusifoliol were individually analyzed by liquid chromatography-mass spectrometry. De novo synthesized sterols were detected as the [13C] labeled sterols. Growth rates were estimated by the ratios of the dry weight of mycelia against drug free control. Results: The MICs of VRCZ against the species were both 0.5 µg/mL. In both species, VRCZ-treated cells showed dosedependent decreases in growth rates and ergosterol biosynthesis rates until almost complete inhibitions. There was little difference between the 80% inhibition concentration of growth rate [IC80 grow] and that of ergosterol biosynthesis rate [IC80 erg] in A. fumigatus (IC80 grow: 0.25 µg/mL, IC80 erg: 0.25 µg/mL). In contrast, IC80 grow was 4 times higher than IC80 erg (IC80 grow: 0.5 µg/mL, IC80 erg: 0.13 µg/mL) in A. flavus. VRCZ treatment showed the accumulation of eburicol and abnormal sterols, and abnormal sterols accumulation per mg-mycelia was similar in both species. In A. fumigatus, the total ergosterol amount per mg-mycelia was not decreased at the IC80 erg, where the mycelial growth was not observed. On the other hand, in A. flavus, it was decreased to the half of the control at the IC80 erg of VRCZ, where the mycelial growth was observed. Conclusions: In vitro VRCZ exposure revealed that the contribution of ergosterol biosynthesis inhibition on the mycelial growth in A. flavus is less than that in A. fumigatus. The ergosterol metabolisms may be different between these two species.
2016

abstract No: 

SUNDAY-432

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)