Engineering a novel probiotic toolkit inEscherichia coli Nissle1917for sensing and mitigating gut inflammatory diseases
Authors: Weibel Nathalie , Curcio Martina , Schreiber Atilla , Arriaga Gabriel , Mausy Marine , Mehdy Jana , Brüllmann Lea , Meyer Andreas , Roth Len , Flury Tamara , Pecina Valerie , Starlinger Kim , Dernič Jan , Jungfer Kenny , Ackle Fabian , Earp Jennifer , Hausmann Martin , Jinek Martin , Rogler Gerhard , Westmann Cauã Antunes 
Published: June 2024

Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacteriumEscherichia coli Nissle 1917(EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide, a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNFα nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to nitric oxide, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by ELISA and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots.

Graphical abstract

Graphical Table of Contents.The engineered probiotic system: Inflamed intestinal cells release the inflammatory regulator TNFα (depicted as red squares), which promotes inflammation through a positive feedback loop. Concurrently, these cells produce large amounts of nitric oxide (NO, represented by yellow circles) during inflammation. Our custom-engineered EcN biosensor can detect NO using a NorR-based sensor (in purple) and subsequently trigger the production of nanobodies (in turquoise). These nanobodies are then released into the extracellular environment via a specially engineered secretion system in the bacterial host (shown in dark blue). Once outside the cell, the nanobodies attach to TNFα, effectively sequestering them and reducing inflammation. The graph at the bottom of this panel illustrates the general behavior of our system: nanobody production starts upon reaching a certain NO concentration threshold and continues in an NO-dependent fashion. As nanobodies are produced, they capture TNFα, leading to a reduction in inflammation and a decrease in NO production. This decrease in NO then halts the nanobody production.

Significance

Probiotics can be engineered to detect and act upon extracellular disease indicators, optimizing therapeutic outcomes. Particularly, self-regulating sense-and-respond genetic circuits have the potential to enhance the accuracy, efficacy, and adaptability of treatment interventions. In this study, we developed and characterized a new integrated and modular toolkit that detects a gut inflammation biomarker, specifically nitric oxide, and responds to it in an inducible manner by secreting humanized nanobodies targeting the pro-inflammatory molecule TNFα. We also develop a coarse-grained mathematical framework for modelling engineered probiotic activity in the gut. This novel system contributes to current efforts to develop new engineered probiotic systems and holds promise for inspiring new treatments for gut inflammation associated with various autoimmune diseases.