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The anti-MRSA compound 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) inhibits protein synthesis in
Nicholas J Carruthers1, Paul M Stemmer2, Joe Media3
1Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48201, USA; Wayne State University, Institute of Environmental Health Sciences, 2309 Scott Hall, 540 E Canfield Ave, Detroit, MI 48202, United States of America.
Abstract:
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× 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 μg/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.
Insights
This study reveals that the natural antibiotic KCR effectively targets Methicillin-resistant S aureus (MRSA) by inhibiting bacterial protein synthesis. Proteomic analysis identified KCR
Area of Science:
- Microbiology
- Proteomics
- Drug Discovery
Background:
- Methicillin-resistant S aureus (MRSA) is a significant cause of patient mortality and prolonged hospital stays.
- 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) is a natural product with demonstrated efficacy against MRSA, but its mechanism of action (MOA) remains unknown.
Purpose of the Study:
- To elucidate the mechanism of action (MOA) of the natural product antibiotic KCR against S aureus using proteomic analysis.
- To identify the specific cellular processes disrupted by KCR in S aureus.
Main Methods:
- Quantitative proteomic analysis was performed on S aureus treated with KCR, oxacillin, or vehicle.
- Isobaric tags and mass spectrometry were employed for protein identification and quantification.
- Time-kill assays determined optimal treatment conditions (4h, 5x MIC) for proteomic analysis.
Main Results:
- Proteomic analysis identified 1190 proteins, with 552 significantly affected by KCR treatment (q < 0.01).
- KCR significantly impacted 6 translation-related categories, including ribosome structure and function, rRNA/tRNA binding, and aminoacyl-tRNA ligase activity.
- KCR demonstrated in vitro inhibition of protein synthesis, confirmed by luciferase inhibition and transcription/translation assays.
Conclusions:
- KCR functions by disrupting S aureus protein synthesis, representing a novel antibiotic mechanism.
- The findings provide crucial insights into KCR's MOA, advancing its development as a potential therapeutic agent against MRSA infections.
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