Nitrate-responsive genetically engineered probiotics locally release TNF-α nanobodies against inflammatory bowel disease.

The clinical potential of oral genetically engineered probiotics is widely recognized, which is usually distinguished by the release of expressed active molecules. However, the impact of the release mode of bacterial payloads remains unknown. Here, we propose a novel release mode, "producing and storing first, then responding and releasing", in a genetically engineered probiotic. Inflammatory bowel disease is a typical chronic intestinal disease, where nitrate in the intestinal route is highly produced. Escherichia coli Nissle 1917 (EcN) was genetically engineered to highly express TNF-α nanobodies (NbTNF-α) for storing and releasing in response to nitrate. Two types of release systems were designed: a protein complex secretion machine, Hly system, and a φX174E-based lysis-release system. These systems were separately recombined into EcN as E5 and E7. The expression and strong anti-inflammation activity of NbTNF-α from E. coli were first confirmed. A NarX/L sensing system was tailored for nitrate, which was a typical biomarker of intestinal inflammation. Both E5 and E7 exhibited similar in vitro kinetics of NbTNF-α secretion. High anti-ulcerative colitis effects were achieved by oral administration of E5 and E7, characterized by inflammation attenuation and recovery of intestinal mucosal barriers, and the levels of NbTNF-α expressed by both in the intestinal tract were similar. This work provides new insights into the design of pathological environment-responsive genetically engineered probiotics against diseases.