Medical Faculty, Institute of Biochemistry and Pathobiochemistry-Microbial Biochemistry, Ruhr University Bochum, Bochum, Germany.
Faculty of Biology and Biotechnology, Applied Microbiology, Ruhr University Bochum, Bochum, Germany.
Microbiol Spectr. 2024 Nov 5;12(11):e0013824. doi: 10.1128/spectrum.00138-24. Epub 2024 Oct 8.
While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism to auranofin when compared to the Gram-positive model organism . The proteome response of exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in 's high tolerance to auranofin. cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether 's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from , which was not the case. However, strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols . Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both and are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to 's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in and other Gram-positive species.
虽然金诺芬对革兰氏阳性菌具有高度的杀菌作用，但由于其机制尚不完全清楚，对革兰氏阴性菌的活性却不显著。在这里，我们旨在阐明革兰氏阴性模式生物 对金诺芬的低敏感性的分子机制，与革兰氏阳性模式生物 相比。暴露于金诺芬的蛋白质组反应表明，含硫酶的失活和全身性氧化应激的诱导相结合。在缺乏金诺芬处理上调蛋白的 突变体中的敏感性测试表明，它们都没有直接参与 的高耐金诺芬性。缺乏外排泵成分 TolC 的 细胞对金诺芬处理更敏感，但敏感程度不足以完全解释革兰氏阳性和革兰氏阴性生物体的敏感性差异。因此，我们测试了 是否 的硫氧还蛋白还原酶（TrxB）比 中的 TrxB 对金诺芬的敏感性更低，事实并非如此。然而，缺乏低分子量巯基谷胱甘肽但不缺乏谷胱甘肽还原酶的 菌株对金诺芬表现出高敏感性。表达遗传编码氧化还原探针 roGFP2 的细菌细胞使我们能够观察到细胞蛋白巯基的氧化。基于我们的发现，我们假设金诺芬导致细菌细胞内巯基氧化还原稳态的全局紊乱，但由于存在药物外排系统和高细胞浓度的谷胱甘肽，革兰氏阴性细菌固有地更具抗性。
意义金诺芬是一种 FDA 批准的治疗类风湿关节炎的药物。然而，它也具有很高的抗菌活性，特别是对革兰氏阳性菌。在当前的抗生素危机中，这将使它成为药物再利用的理想候选药物。然而，它对革兰氏阴性菌的活性低，限制了其广谱应用。在这里，我们表明，在假定的靶标水平上，革兰氏阴性和革兰氏阳性物种之间没有敏感性差异：来自 和 的硫氧还蛋白还原酶都被金诺芬同等抑制。在这两个物种中，金诺芬处理导致全身性氧化蛋白修饰，如蛋白质组学和遗传编码氧化还原探针 roGFP2 监测所示。对金诺芬相对耐药的最大贡献因素似乎是低分子量巯基谷胱甘肽，它在 中不存在，也不存在于其他革兰氏阳性菌中。
