Project Summary Controlling medical device associated infections is an unmet challenge due to high-level tolerance to antibiotics. An important mechanism of such tolerance is the formation of persister cells, which are non- growing phenotypic variants of bacterial cells. Because conventional antibiotics attack bacteria by inhibiting cell growth related behaviors such as cell wall and protein syntheses, they are not effective against persister cells. The capability to escape antibiotic treatment by persister formation and reestablish the bacterial population after treatment is an important intrinsic mechanism of bacterial multidrug tolerance, which leads to chronic infections and facilitates the development of multidrug resistance through acquired mechanisms based on mutations and drug resistance genes. Despite the significance of persister cells, the mechanism of persister formation is still not well understood and control of persister cells remains challenging. One major hurdle to persister research is the lack of an animal model of bacterial persistence that is essential for understanding host response to persister cells and for testing new antimicrobials and biomaterials for persister control. To address this grand challenge, this team will construct the first in vivo model to control and monitor persister formation using blue light. An Escherichia coli strain will be engineered using synthetic biology. Exposure to blue light will induce the toxin gene hipA and repress the antitoxin gene hipB in this strain, leading to high-level persister formation. This process will be reversed by moving cells to the dark, resulting in persister wakeup and reversion to normal cells. Additional components will be included to label all cells with constitutively expressed green fluorescence protein (GFP) and normal cells with bioluminescence expressed under a growth-rate-dependent promoter. After in vitro test, the constructed strain will be validated in an in vivo mouse model of subcutaneous biomaterial infection with an engineered device that generates blue light wirelessly. Persister formation and antibiotic treatment will be tested in this model. The effects will be monitored using whole-animal imaging and the results will be corroborated with microbiological and histopathological analyses after the mice are euthanized. This research team aims to better control persistent infections such as those associated with implanted medical devices. This project will lead to an important millstone toward this ultimate goal by constructing the first animal model of bacterial persistence. With the capability to control and monitor persister formation noninvasively and in real-time, this system will be useful for both fundamental study of bacterial physiology and for drug discovery and biomaterial design against this antibiotic tolerant population. Thus, this project falls well within NIH’s definition of being contributive to "improve people's health and save lives”.