Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules

[Abstract] One of the most compelling approaches in the discovery of novel antimicrobials is screening of natural sources. In our publication we report on the activity of a compound 8hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. We evaluate its applicability for treatment of implant-associated infections. A comprehensive analysis of the mechanism of action of EN4 against Staphylococci revealed its membranolytic properties and a general inhibition of macromolecular biosynthesis, which was confirmed in a macromolecular biosynthesis assay and suggested a multitarget activity. The method used to investigate an influence of EN4 on the synthesis of peptidoglycan, RNA, DNA and proteins is based on precipitation of macromolecules with trichloroacetic acid. These macromolecules are synthesised from respective [ 3 H]-labelled precursors. The incorporated radioactivity with and without an antimicrobial is measured and it reflects the mode of action of the tested compound. Antibiotics with known mechanisms of action are used as controls.