Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin 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 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.

大蒜素（二烯丙基硫代亚磺酸盐）是一种有效的抗菌物质，受伤时由大蒜组织产生，可抵御病原体和害虫。大蒜素是一种活性硫物质（RSS），可氧化谷胱甘肽和蛋白质中的可利用的半胱氨酸。我们使用差异同位素标记法（OxICAT）来鉴定细菌蛋白质组中的大蒜素靶标。在半致死性大蒜素暴露后，我们比较了对大蒜素敏感的荧光假单胞菌Pf 0-1和耐大蒜素的Pf AR-1的蛋白质组。暴露于大蒜素之前，蛋白质主要处于还原状态，大约77％的蛋白质显示不到20％的半胱氨酸氧化。暴露于大蒜素后，蛋白质氧化增加，只有50％来自大蒜素敏感性Pf的蛋白质0-1，但耐大蒜素的Pf AR-1的65％仍少于20％被氧化。DNA促旋酶被鉴定为大蒜素靶标。在未处理的细菌中，DNA促旋酶亚基A（GyrA）中的Cys ₄₃₃大约被氧化了6％。大蒜素处理后，易感Pf 0-1的Cys ₄₃₃氧化度增加到55％，但耐受Pf AR-1的Cys ₄₃₃氧化仅增加到10％。大蒜素在与萘啶酸相同的浓度范围内，在体外抑制大肠杆菌DNA促旋酶活性。与Pf 0-1酶相比，大蒜素在体外对纯化的Pf AR-1 DNA促旋酶的抑制作用更大。替代Pf与Pf 0-1野生型相比，进入Pf 0-1的AR-1 GyrA使交换突变体对大蒜素的敏感性更高。综上所述，这些结果表明，在耐大蒜素的Pf AR-1背景下，GyrA在体内被保护免受氧化，而不是与Pf 0-1 GyrA亚基相比，Pf AR-1 GyrA亚基本来就不容易被大蒜素氧化。DNA促旋酶是具有重要医学意义的抗生素的靶标；因此，大蒜素及其类似物可能有可能单独或与其他治疗剂一起发展为回旋酶抑制剂。