Abstract
Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.
Highlights
The treatment of infectious diseases is and has been one of the main challenges faced by medicine
Our results show that the antimicrobial effect of allicin coincides with a decrease in reduced glutathione levels that is not accompanied by a corresponding increase of glutathione disulfide, S-allylmercapto modification of proteins, and inhibition of metabolic enzymes
Allicin Inhibits Bacterial Growth—To analyze the effect of allicin on the growth of microorganisms, we determined the Minimal inhibitory concentration (MIC) according to Clinical and Laboratory Standards Institute standards on a set of pathogenic bacteria and fungi (Fig. 2A)
Summary
Synthesis of Allicin—Allicin was obtained by oxidation of diallyl disulfide with H2O2 as reported previously with the following modifications [18]. 2 g of diallyl disulfide and 3 ml of 30% H2O2 were dissolved in 5 ml of glacial acetic acid prior to shaking at room temperature for 4 h. Label-free Mass Spectrometric Analysis of the E. coli Proteome after Allicin Exposure—Culture growth, stress treatment, and cell disruption were performed as described for the DTNB assay. After centrifugation (13,000 ϫ g, 30 min, 4 °C) and washing with 5 volumes of acetone, the pellet was resuspended in ammonium bicarbonate buffer and digested with trypsin overnight (37 °C). 100 l of protease inhibitor mixture cOmplete, EDTA-free (one tablet/2 ml of distilled water; Roche Diagnostics) were added prior to cell disruption by ultrasonication (VialTweeter ultrasonicator; 3 ϫ 1 min) at 4 °C. Band intensities were quantified using ImageJ 1.49v [31]
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