Trans-Translation inhibitors and copper 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 statement The 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 CuCl 2 . 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.