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Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography–mass spectrometry

Nature Protocols volume 12, pages 947–963 (2017)Cite this article

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Abstract

Fungal infections and their increasing resistance to antibiotics are an emerging threat to public health. Novel antifungal drugs, as well technologies that can help us bolster the antimicrobial pipeline and understand resistance mechanisms, are needed. The ergosterol biosynthetic pathway is one potential target for antifungal drugs. Here we describe how antifungal susceptibility testing can be combined with target identification in distal ergosterol biosynthesis by means of gas chromatography–mass spectrometry. The fungi are treated with sublethal doses of active components that block ergosterol biosynthesis, and the ergosterol biosynthesis intermediates are analyzed in a targeted metabolomics manner after derivatization (trimethylsilylation). Drug treatment results in distinct sterol patterns that are characteristic of the affected enzyme. Sterol identification based on relative retention times and electron ionization (EI) mass spectra, as well as semiquantitative assessment of ergosterol intermediates, is described. The protocol is applicable to yeasts and molds. The overall analysis time from incubation to test result is not more than 3 d. The assay can be used to determine whether an antifungal compound of interest targets sterol biosynthesis, and, if so, to determine which enzyme in the pathway it targets.

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Figure 1: Main ergosterol biosynthesis pathways under enzyme inhibition.
Figure 2
Figure 3: Separation of sterol isomers obtained after treatment of S. cerevisiae with the morpholine antifungal Fenpropimorph.
Figure 4: Changes in the sterol patterns obtained after incubation of A. fumigatus (mold) (a) and C. krusei (yeast) (b) with the sterol C14-demethylase (enzyme C) inhibitor EMC120B12.

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Acknowledgements

We thank J. Müller for his contribution to the early development of this protocol. Further, we thank EMC microcollections for providing the investigational drug EMC120B12.

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Authors and Affiliations

  1. Department of Pharmacy, Ludwig-Maximilians-University Munich, Munich, Germany

    Christoph Müller, Franz Bracher & Martin Giera

  2. Department of Hygiene, Division of Hygiene and Medical Microbiology, Microbiology and Social Medicine, Medical University Innsbruck, Innsbruck, Austria

    Ulrike Binder

  3. Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden, the Netherlands

    Martin Giera

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Contributions

C.M., U.B. and M.G. carried out the experiments. C.M., F.B. and M.G. designed the protocol. All authors contributed to writing the protocol.

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Correspondence to Martin Giera.

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The authors declare no competing financial interests.

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Müller, C., Binder, U., Bracher, F. et al. Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography–mass spectrometry. Nat Protoc 12, 947–963 (2017). https://doi.org/10.1038/nprot.2017.005

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