Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests.Allicin Leather Flask is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins.We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome.We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure.Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation.
Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised.DNA gyrase was identified as an allicin target.Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria.After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1.Allicin inhibited E.
coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid.Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro Pulley Assembly than the Pf0-1 enzyme.Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type.Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit.DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.