Targeting Multiple Toxin Proteins in Persistent Bacteria with mRNA Display

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Throughout biology, protein functions are critically mediated through interactions with other biomolecules, including with other proteins. The resulting complex array of protein-protein interactions (PPIs) mediates every cellular pathway across all forms of life. A major class of currently untargeted PPIs are toxin-antitoxin interactions which underlie bacterial persistence, a frequent cause of antibiotic treatment failure and relapsing infections. Persistence denotes the ability of bacteria to form persisters, a dormant non-growing phenotype tolerant to antibiotics, and to reinitiate growth after antibiotic removal causing infection relapse.Bacterial toxin-antitoxin (TA) operons, when expressed, form a harmless toxin-antitoxin PPI. Under environmental or antibiotic stress, the antitoxin is rapidly degraded, releasing the toxin which then inactivates bacterial growth, leading to the formation of persisters. Single TA module knockouts in Salmonella have shown markedly reduced levels of persistence. However, multiple TA pairs exist within bacteria, 14 within Salmonella for example, and targeting a single PPI is unlikely to be enough to completely eradicate persisters. Peptide screening technologies are fast emerging as effective methods to rapidly generate high-affinity PPI inhibitors. Of these methods, mRNA display offers the ability to screen libraries of up to 1013 macrocyclic peptides, covering a vast array of chemical space through the introduction of both natural and unnatural amino acids. This method has yet to be employed for the discovery of pan-inhibitors of multiple protein targets.This project will aim to generate pan-toxin inhibitors using mRNA display technology targeting initially three related TA pairs in Salmonella. This work has the potential to increase our ability to prevent persistence and reverse antibiotic tolerance in infectious bacteria.