Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria

Abstract

In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.

Trans-Translation Is an Appealing Target for the Development of New Antimicrobial Compounds

“…Although trans-translation was discovered more than 25 years ago, and has been studied carefully ever since, with several attempts made to develop molecules to target it, the only chemical family that has displayed potential activity derives from 1,3,4-oxadiazole compounds. The recent development of sensitive and selective high-throughput screening assays that target ESKAPE pathogenic bacteria will undoubtedly help us to find new scaffolds that specifically target ribosome rescue [92]. Current studies work from scratch, by screening pharmacologically active small molecules from large chemical or natural product libraries [93], or are based on rational drug design, attempting to target the interactions between tmRNA, SmpB, and the ribosome (Figure 5), as recently described in cryo-EM structural studies [56,57].…”

“…Finally, the most obvious strategy is to use antisense oligonucleotides directed towards the genes encoding SmpB or tmRNA (ssrA gene), or towards the mature tmRNA itself. This approach is already in use in vitro by various laboratories, often as an internal control, with an antisense oligonucleotide targeting the tmRNA MLD and, thereby, very efficiently inhibiting trans-translation (for an example, see [92]).…”

Insights into the ribosomal trans‐translation rescue system: lessons from recent structural studies

“…As the release of non-stop ribosomes is essential for cell survival, and the main rescue system for this is trans-translation, it has to be noted that this process is a very appealing target in the development of new antimicrobial compounds [45,46], especially since it is absent in eukaryotes [47]. Impairing any of the specific interactions that occur between SmpB, tmRNA and the ribosome is therefore a promising strategy for killing pathogenic bacteria or for making them extremely sensitive to existing antibiotics.…”