Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, involves chronic inflammation of the intestine and is associated with impaired mucosal barrier function and dysbiosis of the gut microbiota. Standard therapies (aminosalicylates, antibiotics, corticosteroids, immunosuppressants) often fail to address root causes such as barrier impairment and microbial imbalance and can cause significant adverse effects. Probiotics, including engineered strains that secrete therapeutic cytokines or enzymes, have shown promise for IBD, but challenges remain in achieving sufficient therapeutic levels at disease sites and maintaining probiotic viability through the gastrointestinal tract. The authors hypothesize that engineering Escherichia coli Nissle 1917 (ECN) to overexpress reactive oxygen species (ROS)-scavenging enzymes catalase (CAT) and superoxide dismutase (SOD), combined with a protective chitosan/sodium alginate coating to improve oral delivery and survival, will reduce intestinal ROS, ameliorate inflammation, repair epithelial barriers, and beneficially modulate the gut microbiota in IBD.

The paper situates IBD within a context of barrier dysfunction and microbial dysbiosis, citing work on mucosal barrier integrity and microbiota-derived metabolites. Conventional pharmacologic therapies can induce systemic side effects and do not correct underlying pathophysiology. Prior studies explored natural and engineered probiotics for IBD and other diseases, including engineered ECN to deliver therapeutic domains, and polymeric carriers to enhance probiotic delivery. However, unresolved issues include inadequate therapeutic concentrations in situ and probiotic inactivation by gastric acid and bile salts. Layer-by-layer coatings (e.g., alginate/chitosan) have been reported to protect probiotics and enhance oral delivery. The authors build on this by combining a ROS-scavenging engineered ECN with a biocompatible alginate/chitosan coating to address both efficacy and GI survival.

Engineering and induction: ECN was transformed with pET28a-T5-CAT-SOD to create ECN-PE for stable expression of catalase and superoxide dismutase under IPTG induction (1 mM). Expression was confirmed by western blot. Coating: ECN-PE was coated via layer-by-layer electrostatic assembly using glycol chitosan (2 mg/mL) and sodium alginate (2 mg/mL) in 0.5 M NaCl at pH 6.0. Two alternating layers yielded ECN-PE(C/A)₂. Success of coating was verified by zeta potential shifts (−37.3 mV to +5.9 mV after chitosan; back to −36 to −38 mV after alginate) and increased fluorescence using labeled polymers. Viability after coatings was measured by CCK-8; two layers preserved viability, while three reduced it. Enzyme activity: SOD activity was assessed by O2− scavenging using pyrogallol autoxidation; ECN-PE(C/A)₂ with IPTG achieved ~94% inhibition at 10^7 CFU/mL and showed concentration dependence (10^5–10^7 CFU/mL). CAT activity was evaluated by H2O2 decomposition with dissolved oxygen measurements, showing concentration-dependent O2 generation for induced ECN-PE(C/A)₂. GI protection assays: In vitro survival was tested in simulated gastric fluid (SGF) and 4% bile salts for up to 2 h. Morphology was visualized by TEM. CFUs were enumerated over time. In vivo survival and distribution were assessed using ECN-lux by IVIS 3 h post-gavage and CFU plate counts from stomach, intestine, colon, and cecum at 1, 3, 48, and 72 h. Coating performance was compared to Eudragit L100-55. IBD models and treatment: Acute colitis was induced in female C57BL/6 mice (6–8 weeks) using 3% DSS in drinking water (typically days 0–5/6). Additional models used TNBS or oxazolone following standard sensitization and rectal administration protocols. Treatment groups received PBS, ECN(C/A)₂ (no CAT/SOD), ECN-PE (uncoated), or ECN-PE(C/A)₂, 1 × 10^8 CFU by oral gavage on scheduled days (e.g., days 0, 2, 4, 6 for standard regimen; days 5, 6, 7, 9 for delayed regimen), with 1 mM IPTG in drinking water. Comparators included VSL#3 and Lactobacillus GG. Outcome assessments: Body weight and disease activity index (DAI) were monitored daily. Colon length was measured at necropsy. Intestinal permeability was assessed by FITC-dextran (4 kDa) serum fluorescence. Histopathology (H&E) and blinded damage scoring were performed. Myeloperoxidase (MPO) activity in colon homogenates was quantified. Cytokines (IL-1β, TNF-α, IL-6, IL-10, TGF-β) were measured by ELISA. ROS in colon tissue was visualized by DCFH-DA staining. Tight junction proteins ZO-1 and Occludin were evaluated by immunofluorescence; apoptosis by TUNEL; regeneration by EdU staining. Microbiome analyses: Fecal 16S rRNA gene sequencing (V3-V4) was performed, with OTU clustering (97% identity) and taxonomy via Silva 132 using QIIME 2. Alpha diversity (observed OTUs, Shannon index) and genus-level relative abundance were analyzed. Antibiotic pretreatment (metronidazole, neomycin, vancomycin, ampicillin) was used to deplete microbiota and test dependence of efficacy on microbiome. Safety: Complete blood counts, serum biochemistry (ALT, AST, ALB, BUN), body weight, and major organ histology were assessed after repeated oral dosing.

Coating and activity: Two-layer chitosan/alginate coating successfully assembled on ECN-PE, evidenced by zeta potential alternation and fluorescence increase with layer number. Two layers preserved bacterial viability; a third layer reduced it. Induced ECN-PE(C/A)₂ exhibited strong enzyme function, including ~94% O2− scavenging at 10^7 CFU/mL and robust H2O2 decomposition with concentration-dependent O2 generation. Protection and oral bioavailability: ECN-PE(C/A)₂ maintained morphology after 2 h in SGF or 4% bile (TEM), whereas uncoated ECN-PE showed cell wall damage. In SGF, uncoated ECN-PE rapidly died within 2 h, while ECN-PE(C/A)₂ decreased by only ~1 log10 CFU/mL. In 4% bile, coated bacteria showed significantly improved survival. In vivo, ECN-lux(C/A)₂ bioluminescence in the GI tract was ~5-fold higher than uncoated ECN-lux at 3 h. CFU counts showed ~4-fold more viable coated bacteria in the stomach at 1 h and higher counts in intestine, colon, and cecum at 1, 3, 48, and 72 h. Coating outperformed Eudragit L100-55 in maintaining GI viability. Therapeutic efficacy in DSS colitis: ECN-PE(C/A)₂ significantly mitigated DSS-induced disease versus PBS+DSS and control bacteria, improving body weight trajectories, lowering DAI, preserving colon length, and reducing FITC-dextran permeability. Histology showed reduced damage scores with near-normal epithelium. MPO activity decreased markedly. Pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) were reduced, while anti-inflammatory IL-10 and TGF-β increased. ECN-PE without coating and ECN(C/A)₂ without CAT/SOD were inferior. Barrier and cell effects: ECN-PE(C/A)₂ restored tight junction proteins ZO-1 and Occludin, reduced epithelial apoptosis (TUNEL), and enhanced proliferative regeneration (EdU) compared to DSS alone and other controls. Breadth and comparison: ECN-PE(C/A)₂ outperformed VSL#3 and Lactobacillus GG in DSS colitis and was effective in TNBS- and oxazolone-induced colitis, improving weight, colon length, histology, and inflammation markers. Microbiome modulation and dependence: ECN-PE(C/A)₂ increased alpha diversity (observed OTUs and Shannon index) and shifted composition, elevating butyrate-producing Lachnospiraceae_NK4A136 and Odoribacter, and reducing Escherichia-Shigella in both healthy and DSS-treated mice. Antibiotic pretreatment diminished therapeutic benefits, indicating microbiome involvement. Safety: Repeated oral ECN-PE(C/A)₂ caused no adverse effects; CBC and serum chemistry remained within normal ranges, and organ histology was unremarkable.

The study demonstrates that engineering ECN to produce CAT and SOD, combined with a chitosan/alginate coating, addresses two key barriers to probiotic therapeutics for IBD: ROS-driven inflammation and poor oral survival. The alginate-rich outer layer likely forms a protective skin in acidic and bile environments, improving bioavailability and enabling in situ enzyme activity. In multiple chemical colitis models, ECN-PE(C/A)₂ reduced oxidative stress and inflammatory cytokines, restored tight junctions and barrier function, and promoted epithelial repair. Modulation of the gut microbiota—characterized by increased diversity, enrichment of beneficial butyrate-producing taxa, and reduction of pathogenic Escherichia-Shigella—appears to contribute to efficacy, as suggested by reduced benefits after antibiotic depletion. The platform achieved superior outcomes compared with uncoated or non-expressing controls and common probiotic comparators, while maintaining an excellent safety profile. These findings support a multifunctional mechanism integrating ROS scavenging and microbiome remodeling to ameliorate colitis.

Chitosan/sodium alginate-coated, CAT/SOD-expressing E. coli Nissle 1917 can be effectively delivered orally, survive the GI tract, and exert potent therapeutic effects against acute colitis in mice. The approach reduces ROS and inflammatory mediators, repairs epithelial barriers, and beneficially reshapes the gut microbiota. The strategy showed efficacy across DSS, TNBS, and oxazolone models and was safe upon repeated dosing. This work establishes a platform for living therapeutic protein delivery using engineered probiotics with functional coatings, with potential applicability to other GI and systemic diseases associated with dysbiosis and inflammation. Future work could explore translation to chronic models and clinical settings, optimize induction/regulation of therapeutic expression, and further elucidate host–microbiome interactions underlying efficacy.