The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation.
Summary Here, we present a spatially resolved sampling protocol for the oral human cavity aimed at untargeted metabolomics. We describe the spatial collection of salivary biospecimens, their preparation, and subsequent mass-spectrometry-based untargeted metabolomics analysis. Our protocol avoids complex procedures generally required for gland-specific saliva collection. For the human oral cavity, we provide an easy, flexible, and reproducible solution to comprehensively map the highly heterogeneous environment and elucidate the functionality of salivary components. For complete details on the use and execution of this protocol, please refer to Ciurli et al. (2021) .
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