ИСТИНА

Every year the number of resistant bacterial strains is increasing, therefore searching for the new antimicrobial compounds is a very significant goal of the modern science. Thanks to advances in liquid handling, high-throughput screening of compounds for their antibacterial activity has become a routine procedure. However, defining the mechanism of action (MOA) is still a limiting stage in the characterization of activity. There are many methods, commonly based on luciferase or beta-galactosidase activities and used for specific detection of antibiotic of the certain class. They usually involve some kind of enzymatic reaction and may be carried out only in liquid media, so it is necessary to dilute each substance several times to find the appropriate concentration for the analysis. We designed a method for cost-effective and convenient high throughput screening based on expression of two fluorescent proteins. RFP is induced in the presence of DNA-damaging antibiotics, while Katushka2S is induced by translation stalling agents. This approach was optimized to test up to 586 compounds per single agar plate, without the need in any enzymatic substrates and serial dilutions. By means of this method, we tested 50 thousand individual chemical compounds for antibacterial activity and the mechanisms of action. Several new translation inhibitors were found. Their MOA was confirmed by in vitro experiments.