PROJECT SUMMARY Chronic bacterial infections are difficult to treat through standard antibiotics because a small populationof the bacteria becomes tolerant to antibiotics by entering a dormant state. Such bacteria are known as persistersand pose a significant problem in clinical settings. As antibiotic levels drop post-treatment, persister cells revertback into actively growing cells resulting in recalcitrant chronic infections. Currently, there are very limitedapproaches available for eradicating persister cells. We propose leveraging antibacterial protein colicins as ananti-persister agent. Colicins kill cells physically by disrupting cell envelopes and degrading DNA and RNA.Colicins are excellent candidates for killing persisters because the mechanisms of colicin action are growth-independent. We will utilize cell-free protein synthesis (CFPS) to produce colicins, as CFPS offers rapid proteinproduction and characterization of such toxic proteins without cell-viability constraints. The goal of this project is to investigate and engineer colicins to control non-growing persister cells fortherapeutic applications. The proposed research will be advanced with three integrated research Aims. In Aim1, we will characterize the effects of different types of colicins on persister cells to get insights on what kind ofcell killing mechanisms enable colicins to eradicate persister cells. Colicins have great potential to be developedinto target-specific therapeutics without affecting beneficial bacteria. In Aim 2, we will evolve colicins via directedevolution to have enhanced activity. The engineered colicins will be identified through a fluorescence-basedhigh-throughput screening approach using CFPS. We anticipate developing highly active and target-specificcolicins that can be applied for treating persistent infections. In Aim 3, we will investigate redirecting the activityof colicins toward other pathogens which are not recognized by native colicins. The chimeric colicins will beconstructed by replacing domain proteins of colicins with those of colicin-like proteins from other bacteria. Ourresearch will open the development of customized anti-persister agents that can selectively kill antibiotic-tolerantpathogens.