Engineered Escherichia coli Nissle 1917 secreting anti-TNF-α nanobody as a single-strain live biotherapeutic for inflammatory bowel disease.

The rising global incidence of inflammatory bowel disease (IBD) creates an urgent need for safer, gut-targeted therapies. Current treatments, from small-molecule drugs to systemic anti-tumor necrosis factor-alpha (TNF-α) biologics, are frequently limited by off-target immunosuppression, heightened infection risk, and poor mucosal bioavailability. Engineered probiotic-based live biotherapeutics offer a compelling alternative by enabling localized drug production within the inflamed intestine. We engineered Escherichia coli Nissle 1917 (EcN) to secrete the anti-TNF-α nanobody MT1, creating the streamlined, single-strain platform EcN-MT1. Five signal peptides were screened, and plasmid-based and CRISPR-Cas9-mediated chromosomal integration strategies were compared. Structural modeling and molecular dynamics simulated MT1-murine TNF-α (mTNF-α) binding. Binding affinity and anti-inflammatory activity were assessed by ELISA and in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Therapeutic efficacy was further evaluated in a dextran sulfate sodium (DSS)-induced murine colitis model by assessing body weight, disease activity index (DAI), colon length, histopathology, colonic pro-inflammatory cytokines, and 16S rRNA gut microbiota profiling. Among the tested signal peptides, α-hemolysin (HlyA) achieved highest secretion (4.6 mg/L), and the plasmid-based strain markedly outperformed genomic integrants without impairing growth. Simulations confirmed stable complementarity-determining regions (CDR)-mediated binding, consistent with the high affinity (EC50 27.9 nM) and potent suppression of LPS-induced mRNA expression of Tnf and interleukin-1β (Il1b) in macrophages. In the DSS-induced murine colitis model, oral administration of EcN-MT1 significantly attenuated weight loss, improved DAI scores, and preserved colon length. Histopathological analysis revealed reduced mucosal ulceration, crypt loss, and immune cell infiltration, accompanied by downregulated colonic Tnf and Il1b mRNA. Notably, EcN-MT1 treatment restored gut microbial diversity, corrected dysbiosis, and enriched beneficial taxa linked to butyrate production, barrier enhancement, and anti-inflammatory effects. This study establishes EcN-MT1 as a potent, orally deliverable live biotherapeutic that achieves localized TNF-α neutralization while concurrently promoting microbial and mucosal homeostasis, offering a novel and translatable strategy for IBD treatment.