Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

The rising prevalence and severity of antibiotic-resistant biofilm infections poses an alarming threat to public health worldwide. Here, biocompatible multi-compartment nanocarriers were synthesized to contain both hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and the hydrophilic antibiotic methicillin for the treatment of medical device-associated infections. SPION co-encapsulation was found to confer unique properties, enhancing both nanocarrier relaxivity and magneticity compared to individual SPIONs. These iron oxide-encapsulating polymersomes (IOPs) penetrated 20 μm thick Staphylococcus epidermidis biofilms with high efficiency following the application of an external magnetic field. Three-dimensional laser scanning confocal microscopy revealed differential bacteria death as a function of drug and SPION loading. Complete eradication of all bacteria throughout the biofilm thickness was achieved using an optimized IOP formulation containing 40 μg/mL SPION and 20 μg/mL of methicillin. Importantly, this formulation was selectively toxic towards methicillin-resistant biofilm cells but not towards mammalian cells. These novel iron oxide-encapsulating polymersomes demonstrate that it is possible to overcome antibiotic-resistant biofilms by controlling the positioning of nanocarriers containing two or more therapeutics.

Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.

30There is an unmet need for new treatment methods for inflammatory bowel disease (IBD) that 31 can reliably maintain remission without leading to detrimental side effects. Beneficial bacteria 32 have been utilized as an alternative treatment for IBD albeit with low efficacy. We genetically 33 engineered Escherichia coli Nissle 1917 (EcN) to create an anti-inflammatory fibrous matrix in 34 situ. This matrix consists of EcN-produced curli nanofibers displaying trefoil factors (TFFs), 35 known to promote intestinal barrier function and epithelial restitution. We confirmed that 36 engineered EcN was able to secrete the curli-fused TFFs in vitro and in vivo, and was non-37 pathogenic. We observed an enhanced protective effect of engineered EcN against dextran 38 sodium sulfate induced colitis in mice, associated with barrier function reinforcement and 39 immunomodulation. This work sets the foundation for the development of a novel therapeutic 40 platform in which the in situ production of a therapeutic protein matrix from beneficial bacteria 41 can be exploited. 42 90 concentrations of the therapeutic molecule at the site of disease. Indeed, concerns have been 91 raised about the compatibility of this strategy with immunomodulatory biologics, since the 92 mucosal epithelial barrier hinders their trafficking to their target cells in the lamina propria (13, 93 14). Nevertheless, the promise of effective treatments that can be produced cheaply, delivered 94 orally, and minimize systemic side effects has continued to fuel interest in microbes as 95 therapeutics. 96Here we present an alternative approach to engineered microbial therapies for IBD 97 treatment. Instead of secreting soluble therapeutic proteins, we programmed bacteria to assemble 98 a multivalent material decorated with anti-inflammatory domains in the gut. The displayed 99 domains are designed to target the material to the mucosal layer of the epithelium and promote 100 host processes that reinforce epithelial barrier function ( Figure 1). The bacterially-produced 101 scaffold for the living material is based on curli fibers, a common proteinaceous component of 102 bacterial extracellular matrices. Hence, we refer to our approach as Probiotic Associated 103 Therapeutic Curli Hybrids (PATCH). We demonstrate that PATCH is capable of ameliorating 104 inflammation caused by dextran sodium sulfate (DSS) induced colitis in a mouse model. 105 Results 106We used E. coli Nissle 1917 (EcN) as our cellular chassis for PATCH. EcN is well-studied, has a 107 long track record of safety in humans, and is a popular starting point for engineered therapeutic 108 microbe efforts because of its compatibility with canonical genetic engineering techniques for 109 bacteria (15). In addition to its use as an over-the-counter supplement for general GI disorders, 110 EcN has also been evaluated in comparison to mesalazine for maintaining remission in ulcerative 111 colitis in randomized control trials (16). While EcN lead to some favorable outcomes, overall 112 efficacy...