Vazquez-Hernandez, M. | Search bioRxiv

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2023

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

The antirheumatic gold(I) compound auranofin has been suggested to exhibit bactericidal activity by disrupting the thiol homeostasis through direct inhibition of bacterial thioredoxin reductase (TrxB). While highly effective at killing Gram-positive bacteria, it 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 Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. A change in the proteome of E. coli exposed to auranofin suggested that the effect of this gold compound is a combination of inactivation of thiol-containing enzymes, upregulation of proteins involved in basal metabolism, and the consequent induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking the proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. colis high tolerance to auranofin. To elucidate factors that could make Gram-negative bacteria less susceptible to auranofin, we tested E. coli cells lacking the efflux pump component TolC. These cells were more sensitive to auranofin treatment than the wild type, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested if E. colis thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. E. coli cells lacking components of the thioredoxin-system were also only marginally more susceptible to auranofin. However, E. coli 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 in situ. In line with their susceptibility, the kinetics of probe oxidation and the degree of oxidation promoted by auranofin is significantly higher in Gram-positive bacteria when compared to Gram-negative bacteria. 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.

biochemistry↗