(469h) Antimicrobial Mechanism of Resveratrol-Trans-Dihydrodimer Produced From the Enzymatic Oligomerization of Resveratrol Mediated By Soybean Peroxidase

Many natural polyphenols possess low antimicrobial activities which can be improved by using them as scaffolds to generate libraries of their oligomers. We have performed the in vitro diversification of resveratrol mediated by soybean peroxidase, and isolated two dimers (resveratrol-trans-dihydrodimer, and pallidol) and tested them as antimicrobials. In particular, resveratrol-trans-dihydrodimer was found to be an effective antimicrobial against Gram positive strains B. cereus (MIC = 15.0 µM), Listeria (125.0 µM), and S. aureus (MIC = 62.0 µM) as well as against Gram negative E. coli (MIC = 122.5 µM; in the presence of efflux pump inhibitor Phe-Arg-β-naphthylamide), while pallidol did not show significant antimicrobial activity. Further we deciphered the mechanism of action of the trans-dihydrodimer by transcriptomic, biochemical and flow cytometry analysis. Gene expression microarray and qRT-PCR data from B. cereus indicated down regulation of ABC transporters (ATP binding proteins). Flow cytometry analysis showed a strong depolarization of cell membrane upon treatment with resveratrol-trans-dihydrodimer, while ATP measurement indicated an accumulation of overall ATP (>2 fold); down regulation of ABC transporters that break down ATP is one of the individual events that contributes for ATP accumulation.

Alternately, a decrease in nucleic DNA content of B. cereus and E. coli upon incubation with the resveratrol-trans-dihydrodimer was also observed by flow cytometry. An in vitro activity assay of DNA gyrase from E. coli showed a strong inhibitory effect caused by the resveratrol-trans-dihydrodimer, which is consistent with previous reports regarding the inhibitory effect of polyphenols on DNA gyrase activity. This enzyme requires ATP as a source of energy for supercoiling DNA and its inhibition stops the breakdown of ATP. This observation suggests that resveratrol-trans-dihydrodimer could be responsible for inhibiting other enzymes that require ATP resulting in the increase of overall ATP. Finally, we have demonstrated that the enzymatic oligomerization of resveratrol is an attractive strategy for generating effective antimicrobials with potential therapeutic application especially due the low toxicity of resveratrol derivatives against mammalian cells.