Co-translational protein aggregation and failed ribosome rescue as a novel broad-spectrum antibacterial mechanism

Drug-resistant bacteria pose an urgent global health threat, necessitating the development of antibacterial compounds with a novel mode of action. Protein biosynthesis accounts for up to half of the energy expenditure of bacterial cells and consequently inhibiting the efficiency or fidelity of the bacterial ribosome is a major target of existing antibiotics. Here we describe an alternative mode of action to affect the same process; allowing translation to proceed but causing co-translational aggregation of the nascent chain. We found a peptide that acts via this mechanism and is bactericidal against a wide range of pathogenic bacteria, including strains for which novel treatments are most urgently needed such as the ESKAPE pathogens, both in planktonic growth and in biofilms. Proteomics, transcriptomics and biochemical analyses of the inclusion bodies that form in bacteria upon peptide treatment reveal ribosomal proteins, mRNAs and their cognate nascent chains in amyloid-like structures assembled in polar inclusion bodies. The bacterial ssrA ribosome rescue pathway is activated but fails to prevent incorporation of hundreds of proteins into the inclusion bodies. Mechanistically, our results show that disrupting ribosomal quality control via co-translational aggregation constitutes a potent target mechanism for the development of broad-spectrum antibacterials. Overall design: To investigate the molecular mechanisms underlying inclusion body formation and proteostatic collapse, we began by conducting RNA sequencing (RNA-seq) on bacterial strains treated with P33, a compound known to promote protein aggregation. Additionally, we treated another set of bacteria at 49°C as a positive control for protein aggregation. To minimize variability and ensure robust statistical analysis, five biological replicates were performed for each condition.