Cytometric proteome profiling of GFP-tagged yeast for characterizing novel antifungals
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.
Authors
- Vitaly V. Kushnirov (ORCID: https://orcid.org/0000-0003-0316-0766)
- Victoria Bidiuk (ORCID: https://orcid.org/0000-0003-0883-7174)
- Darya A. Avdanina (ORCID: https://orcid.org/0000-0003-1501-5865)
- I. B. Levshin (ORCID: https://orcid.org/0000-0003-1024-9099)
- Dmitry A. Makarov (ORCID: https://orcid.org/0000-0003-2438-1146)
- Olga V. Mitkevich (ORCID: https://orcid.org/0000-0002-1932-9979)
- Alexander I. Alexandrov (ORCID: https://orcid.org/0000-0001-7250-4740)
- Alexander A. Zhgun (ORCID: https://orcid.org/0000-0002-8672-8638)
- Olga Riabova (ORCID: https://orcid.org/0000-0002-3794-6495)
- Maxim V. Jasko (ORCID: https://orcid.org/0000-0003-0462-8930)
- Michael O. Agaphonov (ORCID: https://orcid.org/0000-0002-9270-9476)
- Liudmila A. Alexandrova (ORCID: https://orcid.org/0000-0002-8929-4898)
- Anna Egorova (ORCID: https://orcid.org/0000-0002-6708-2421)
- Anna A. Ermolyuk (ORCID: https://orcid.org/0009-0009-9780-7954)
- Fedor Ryabov
- Yaroslav M. Stanishevskiy
- Eslam Ghazy
Institutions
- Engelhardt Institute of Molecular Biology (RU)
- University of Geneva (CH)
- National Research University Higher School of Economics (RU)
- Peoples' Friendship University of Russia (RU)
- Tanta University (EG)
- Federal Research Centre of Nutrition and Biotechnology (RU)
- Development Agency of Serbia (RS)
- Gause Institute of New Antibiotics Russian Academy of Medical Sciences (RU)
- Scientific Research Institute of Technology named after AP Aleksandrov (RU)
- Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences (RU)
- Weizmann Institute of Science (IL)
Publication Details
- Journal
- Communications Biology
- Published
- 2026-04-27
- DOI
- https://doi.org/10.1038/s42003-026-10083-5
- Primary Topic
- Click Chemistry and Applications
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Université de Genève
- Russian Science Foundation
- Ministry of Science and Higher Education of the Russian Federation