Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

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 <i>Escherichia coli</i> to auranofin when compared to the Gram-positive model organism <i>Bacillus subtilis</i>. The proteome response of <i>E. coli</i> exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in <i>E. coli</i> mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in <i>E. coli</i>'s high tolerance to auranofin. <i>E. coli</i> 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 <i>E. coli</i>'s thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from <i>B. subtilis</i>, which was not the case. However, <i>E. coli</i> 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 <i>in situ</i>. 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 <i>Escherichia coli</i> and <i>Bacillus subtilis</i> 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 <i>E. coli</i>'s relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in <i>B. subtilis</i> and other Gram-positive species.