(188ca) Colicin Production Using Cell-Free Protein Synthesis to Control Persister Cell Formation

Persister cells are metabolically dormant bacterial populations that are frequently found in biofilms and increased under environmental stresses. Since persister cells are not growing, they can survive under very high concentrations of conventional antibiotics that can kill actively growing cells, which has been a serious problem in treating chronic bacterial infections. Therefore, there is an urgent and growing need for developing novel antimicrobial drugs that can kill non-growing persister cells. Colicins, a type of antimicrobial bacteriocins, are considered as a viable alternative of conventional antibiotics due to their unique cell killing mechanisms that can damage cells by pore-forming on the cell membrane, nuclease activity, and cell wall synthesis inhibition. Additionally, colicins’ cell killing activity is target-specific by recognizing receptors of the target cells without influencing other bacteria. However, recombinant production of colicins requires co-production of immunity protein to protect host cells, otherwise the colicins are lethal to the host. To maximize the colicin production and avoid the cell viability concerns without co-production of immunity protein, we utilized cell-free protein synthesis to produce colicins with different modes of action. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Then, we examined to see if colicins can kill persister cells upon antibiotics treatment. Colicin E1 and E2 effectively killed persister cells prepared from both exponential as well as stationary phase cells by treating with Ciprofloxacin antibiotic. This study supports that colicins can be developed to kill persister cells that are responsible for chronic diseases, and cell-free protein synthesis can be used as a promising platform for rapid production and characterization of toxic proteins.