Probiotic Associated Therapeutic Curli Hybrids (PATCH)

Abstract

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...

Robust Self‐Regeneratable Stiff Living Materials Fabricated from Microbial Cells

“…In these examples, cells were either combined with synthetic polymers or biopolymers to create composites, or they were designed/selected to produce a specific extra‐cellular matrix (e.g., curli fibers, cellulose). [ 15–30 ] In our recent work, we have shown that this approach can be used to fabricate macroscopic stiff (2‐4 GPa) thin films by exploiting the stiff structural characteristics of curli fibers. [ 31 ] In contrast to the above approaches, we wondered whether living cells alone, without any extracellular matrix, could serve as the primary building block in ELM fabrication and thereby incorporate aspects of self‐regeneration.…”

“…[15,16] Additional work has focused on ELMs that function as catalytic surfaces, filtration membranes, under-water adhesives, pressure sensors, conductive films, gut adhesives, etc. [17][18][19][20][21][22][23][24][25][26][27][28][29][30] In spite of rapid progress in the field, it should be noted that there are few examples of ELMs that combine the structure-building capabilities of cells with their other capabilities, like self-regeneration, self-healing, or environmental responsiveness. ELM technologies that can streamline fabrication by relying more on autonomous cellular functions could help advance the field from a fundamental perspective and lead to ELMs compatible with scalable manufacturing techniques.…”

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

“…To demonstrate the potential functionality of the capsules, we produced hybrid capsules containing engineered curli nanofibers displaying various functional protein domains. Curli are insoluble and robust functional protein fibers anchored to the surface of E. coli cells, whose versatility as a platform for ELMs has been explored in many applications, including adhesion to abiotic surfaces, [ 16 ] in vivo display of therapeutic domains, [ 45 ] and sequestration of pathogens from drinking water, [ 46 ] among others. [ 47 ] We hypothesized that we could add a range of functionality to the capsules by expressing curli‐tethered proteins, which would remain fixed within the BC matrix while interacting with the external solution.…”

Chemically and Biologically Engineered Bacteria‐Based Delivery Systems for Emerging Diagnosis and Advanced Therapy

“…developed an engineered EcN that generated fibrous matrices to promote mucosal healing for alleviating IBD. [ 118 ] Curli fiber proteins were fused with trefoil factor (TFF) domains to promote epithelial restitution, and the EcN was engineered to create fibrous matrices through the in situ self‐assembly of the modified curli fibers ( Figure A–D). The results demonstrated that the engineered EcN significantly reduced the weight loss of mice, decreased pro‐inflammatory cytokine levels, prevented colon length reduction, and displayed superior anti‐inflammation efficacy in a dextran sodium sulfate (DSS)‐induced colitis model.…”