Allicin induces thiol stress in bacteria through S-allylmercapto modification of protein cysteines

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.