(135g) Invited Speaker: Eradicating Antibiotic-Tolerant Bacteria and Biofilms By Antimicrobial Modular Proteins Produced By Cell-Free Protein Synthesis

Chronic bacterial infections are difficult to treat through standard antibiotics because a small population of the bacteria in biofilms becomes tolerant to antibiotics by entering a dormant state. Such antibiotic tolerant bacteria are known as persisters and pose a significant problem in clinical settings. As antibiotic levels drop post-treatment, persister cells revert back into actively growing cells resulting in recalcitrant chronic infections. Currently, there are very limited approaches available for eradicating 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. Thus, colicins are excellent candidates for killing persisters. We utilized cell-free protein synthesis (CFPS) to produce colicins, as CFPS offers rapid protein production and characterization of such toxic proteins without cell-viability constraints and the need for protein purification. We demonstrated that colicins (e.g., E1 and E2) produced by CFPS based on crude Escherichia coli lysates are active in eradicating persister cells by up to six orders of magnitude of cell viability reduction. Optimizing CFPS reactions such as including molecular chaperones and immunity proteins enabled significant improvement of solubility and activity of colicins. Colicin treatment was also effective in killing biofilm cells. Colicin production in the CFPS setting and cell-killing kinetics of cell-free synthesized colicins were very rapid, which provides a great potential for point-of-care drug producing systems by combining with lyophilized or paper-based CFPS. We further discuss the modular design of colicins that can redirect activity of colicins to target other bacteria and developing beneficial microbes that can eradicate or kill harmful biofilm cells via controlled colicin secretion. This study finds that colicins can eradicate antibiotic-tolerant bacteria and biofilm cells and that CFPS is a promising platform for rapid production, optimization, and characterization of toxic proteins.