TOWARDS A GENETICALLY-ENGINEERED BACTERIUM FOR GASTROINTESTINAL WOUND HEALING

Abstract

Society and physicians frequently associate the increase of antibiotic-resistant bacteria with the overuse of antibiotics. This proposes a question, “Why use antibiotics to fight bacteria and risk resistance, when one could engineer bacteria to target and kill infectious bacteria?” Bacteria are often thought of as ‘good’ bacteria (e.g., commensals, probiotics) or ‘bad’ bacteria (e.g., pathogens). Synthetic biology enables the augmentation of biosynthetic capabilities and retooling of regulatory structures in the creation of cells with unprecedented ability to make products. One can also, however, think of the cell as the product – a cell that operates in a noisy environment to execute non-native tasks. There have been several recent reports of the rewiring of bacterial cells to function as conveyors of therapeutics. The engineering and rewiring of the bacteria such as E. coli into ‘smart' bacteria potentially allows for a broad range of applications, from the treatment of wounds, the elimination of pathogenic strains, to the delivery of vaccines, particularly in the gastrointestinal (GI) tract. I have engineered smart bacteria as a therapeutic delivery vehicle for wound healing in the GI tract. The approach comprises synthetic biology and microfluidics for the creation of a biological ‘test track' for ensuring the appropriate design and testing of engineered bacteria. Bacterial motility was engineered for response to wound-generating signals such as hydrogen peroxide. Specifically, we have placed a motility enzyme CheZ under the control of the hydrogen-peroxide-responsive oxyR/S gene-promoter pair so that the ‘run’ in the tumble and run scheme of bacterial movement is externally regulated. These engineered cells exploit pseudotaxis for directional swimming towards hydrogen peroxide, a non-native signal. Additionally, the therapeutic enzyme transglutaminase plays an important role in the tissue clotting cascade. Microbial transglutaminase can crosslink fibrinogen, similar in function to human transglutaminases during the clotting cascade, but independently of calcium ions. This allows for a potentially faster, increased wound-healing response. By combining microbial transglutaminase expression with controlling motility and lysis expression using the OxyR/S system, the ‘smart’ bacteria can potentially swim towards and treat at the wound site with subsequent cell lysis. Ultimately, this strategy can lead to new bacterial therapies.