title: Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography-mass spectrometry.
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.
authors.affiliation: Department of Pharmacy, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Müller
authors.affiliation: Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Binder
authors.affiliation: Department of Pharmacy, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Bracher
authors.affiliation: Department of Pharmacy, Ludwig-Maximilians-University Munich, Munich, Germany.|Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden, the Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Giera
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D053898:Biosynthetic Pathways / Q000187:drug effects*; D004875:Ergosterol / Q000096:biosynthesis*; D005658:Fungi / Q000737:chemistry* / Q000187:drug effects*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods*
chemical_list: D000935:Antifungal Agents; D004875:Ergosterol