Sterol Composition of Clinically Relevant Mucorales and Changes Resulting from Posaconazole Treatment

Abstract

Mucorales are fungi with increasing importance in the clinics. Infections take a rapidly progressive course resulting in high mortality rates. The ergosterol biosynthesis pathway and sterol composition are of interest, since they are targeted by currently applied antifungal drugs. Nevertheless, Mucorales often exhibit resistance to these drugs, resulting in therapeutic failure. Here, sterol patterns of six clinically relevant Mucorales (Lichtheimia corymbifera, Lichtheimia ramosa, Mucor circinelloides, Rhizomucor pusillus, Rhizopus arrhizus, and Rhizopus microsporus) were analysed in a targeted metabolomics fashion after derivatization by gas chromatography-mass spectrometry. Additionally, the effect of posaconazole (POS) treatment on the sterol pattern of R. arrhizus was evaluated. Overall, fifteen different sterols were detected with species dependent variations in the total and relative sterol amount. Sterol analysis from R. arrhizus hyphae confronted with sublethal concentrations of posaconazole revealed the accumulation of 14-methylergosta-8,24-diene-3,6-diol, which is a toxic sterol that was previously only detected in yeasts. Sterol content and composition were further compared to the well-characterized pathogenic mold Aspergillus fumigatus. This work contributes to a better understanding of the ergosterol biosynthesis pathway of Mucorales, which is essential to improve antifungal efficacy, the identification of targets for novel drug design, and to investigate the combinatorial effects of drugs targeting this pathway.

Keywords: Mucorales; Rhizopus arrhizus; antifungal effectivity; gas chromatography-mass spectrometry (GC-MS); posaconazole; sterol pattern.

Figure 1

Preferred ergosterol biosynthesis pathways in yeasts (S. cerevisiae) and molds (A. fumigatus) starting from lanosterol. Enzymes: (A) sterol C14-demethylase, (B) sterol C24-methyltransferase.

Figure 2

Representative selected ion chromatograms of untreated R. arrhizus samples (blue) and hyphae confronted with posaconazole (red). Hyphae were confronted with sublethal concentrations (0.5 µg/mL) for 4 h. Numbers in the diagram represent the sterol intermediates as given in Table 1. X-axis presents retention time. Selected ions for chromatogram (A) m/z 363 + 366 + 365 + 407 + 408 and selected ions for chromatogram (B) m/z 343 + 377 + 379 + 393 + 472 + 486.

Figure 3

Relative amounts [%] of the most prominent sterols in posaconazole treated (red) and untreated (blue) R. arrhizus hyphae. Cultures, pre-grown for 16 h, were confronted with 0.5 µg/mL posaconazole for 4 h before sterol extraction. Sterol pattern was compared to untreated controls, which were incubated under identical conditions. Error bars represent standard deviation out of two independent experiments, comprising six technical replicates. (* p < 0.05; *** p < 0.001: student’s t-test). For detailed information on all sterols extracted, see Table 2.

Figure 4

Putative alternative sterol biosynthesis pathway from lanosterol in Mucorales in the presence of posaconazole. Enzymes: (A) sterol C14-demethylase, (B) sterol C24-methyltransferase, (C) sterol C4-demethylase complex, (D) sterol C5-desturase.