The anti-MRSA compound 3-O-alpha-L-(2",3"-di-p-coumaroyl)rhamnoside (KCR) inhibits protein synthesis in Staphylococcus aureus

Methicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L(2 '',3 ''-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). We used proteomics to identify the MOA for KCR. Methicillin sensitive S aureus and a mixture of four KCR stereoisomers were tested. A time-kill assay was used to choose a 4 h treatment using KCR at 5 x its MIC for proteomic analysis. S aureus was treated in triplicate with KCR, oxacillin or vehicle and quantitative proteomic analysis was carried out using isobaric tags and mass spectrometry. 1190 proteins were identified and 552 were affected by KCR (q < 0.01). Ontology analysis identified 6 distinct translation-related categories that were affected by KCR (PIANO, 10% false-discovery rate) including structural constituent of ribosome, translation, rRNA binding, tRNA binding, tRNA processing and aminoacyl-tRNA ligase activity. Median fold changes (KCR vs Control) for small and large ribosomal components were 1.46 and 1.43 respectively. KCR inhibited the production of luciferase protein in an in vitro assay (IC50 39.6 mu g/ml). Upregulation of translation-related proteins in response to KCR indicates that KCR acts to disrupt S aureus protein synthesis. This was confirmed with an in vitro transcription/translation assay. Significance: Methicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L-(2 '',3 ''-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). Using proteomic analysis we determined that KCR acts by inhibiting protein synthesis. KCR is an exciting novel antibiotic and this work represents an important step in its development towards clinical use.