Cytometric proteome profiling of GFP-tagged yeast for characterizing novel antifungals

Communications Biology. 2026. Vol. 9. Article 885.

Ghazy E., Bidiuk V., Ryabov F., Mitkevich O., Riabova O., Stanishevskiy Y., Levshin I.

Assaying cellular responses to antimicrobial molecules is one way to understand modes of action of potential drugs. This is often achieved via transcriptomics and proteomics, but simple, inexpensive methods for rapid characterization are lacking. To bridge this gap, we assayed changes in the abundance of a panel of 64 “sentinel” proteins fused to GFP in the yeast Saccharomyces cerevisiae using flow cytometry. This method produced expected patterns for classical antifungals and allowed inference of common mechanisms between known and novel compounds. Single-cell data also revealed diverging responses in mitochondrial protein abundance in response to thiazolidine antifungals, and perturbations of the cell cycle caused by various compounds. Finally, the method provided insight into the unknown mode of action of alkylated nucleosides, which can be used against fungi residing on works of art. These substances elevate levels of proteins involved in the biosynthesis of aromatic amino acids (AAA), as well as in oxidative stress. Furthermore, deficiencies of Trp and Tyr biosynthesis increased the efficacy of these compounds, while antioxidants reduced it. Most surprisingly, antioxidant effectiveness relied on AAA biosynthesis. Thus, our approach and its possible modifications for other microbes provide an easy and reliable platform for revealing modes of action of novel compounds.

Keywords: proteome, протеом, flow cytometry, флуоресцентные белки, yeast, проточная цитометрия, дрожжи, GFP

Publication based on the results of: Development of the concept of a genetic computer (2025)