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

Abstract

Reactive oxygen species (ROS) play vital roles in intestinal inflammation. Therefore, eliminating ROS in the inflammatory site by antioxidant enzymes such as catalase and superoxide dismutase may effectively curb inflammatory bowel disease (IBD). Here, Escherichia coli Nissle 1917 (ECN), a kind of oral probiotic, was genetically engineered to overexpress catalase and superoxide dismutase (ECN-pE) for the treatment of intestinal inflammation. To improve the bioavailability of ECN-pE in the gastrointestinal tract, chitosan and sodium alginate, effective biofilms, were used to coat ECN-pE via a layer-by-layer electrostatic self-assembly strategy. In a mouse IBD model induced by different chemical drugs, chitosan/sodium alginate coating ECN-pE (ECN-pE(C/A)₂) effectively relieved inflammation and repaired epithelial barriers in the colon. Unexpectedly, such engineered EcN-pE(C/A)₂ could also regulate the intestinal microbial communities and improve the abundance of Lachnospiraceae_NK4A136 and Odoribacter in the intestinal flora, which are important microbes to maintain intestinal homeostasis. Thus, this study lays a foundation for the development of living therapeutic proteins using probiotics to treat intestinal-related diseases.

Fig. 1. Engineered probiotics for IBD treatment.

a ECN was genetically engineered to produce CAT and SOD, which can scavenge ROS efficiently. Then, the engineered ECN was encapsulated by chitosan and sodium alginate through the layer-by-layer strategy (ECN-pE(C/A)₂). ECN Escherichia coli Nissle 1917; ECN-pE, ECN(pET28a-T5-CAT-SOD). b ECN-pE(C/A)₂ was orally delivered to the mice, and its bioavailability within the GI tract was significantly elevated with chitosan/sodium alginate coating. c ECN-pE(C/A)₂ exerts a prominent palliative effect to relieve inflammatory bowel disease by eliminating ROS and regulating the intestinal flora.

Fig. 2. Characterization of ECN-pE(C/A)2.

a Zeta potential of ECN-pE with different chitosan/sodium alginate layer coatings during the preparation of ECN-pE(C/A)₂. b Fluorescence intensity of ECN-pE with different fluorescence-labeled chitosan or sodium alginate layers. Chitosan was labeled with Cy5.5, and sodium alginate was labeled with FITC. a.u. arbitrary unit. c The relative viabilities of ECN-pE before and after different coatings as indicated. d Western blot analysis of the expression of CAT and SOD. CAT catalase, SOD superoxide dismutase. A representative western blot image from two independent experiments. e The ·O₂− inhibition rate of ECN(C/A)₂(−), ECN(C/A)₂(+), ECN-pE(C/A)₂(-) and ECN-pE(C/A)₂(+) at the same concentration of 10⁷ CFU/mL. ECN(C/A)₂(−): ECN(C/A)₂ without 1 mM IPTG induction, ECN(C/A)₂(+): ECN(C/A)₂ with 1 mM IPTG induction, ECN-pE(C/A)₂(−): ECN-pE(C/A)₂ without 1 mM IPTG induction, ECN-pE(C/A)₂(+): ECN-pE(C/A)₂ with 1 mM IPTG induction. f The ·O₂− inhibition rate of ECN-pE(C/A)₂(+) at different concentrations (10⁵–10⁷ CFU/mL). g The oxygen generation capacity of ECN(C/A)₂(−), ECN(C/A)₂(+), ECN-pE(C/A)₂(−), and ECN-pE(C/A)₂(+) in H₂O₂ solutions with the same concentration of 5 × 10⁸ CFU/mL. h The oxygen generation capacity of ECN-pE(C/A)₂(+) with different concentrations of ECN-pE(C/A)₂ (10⁷–5 × 10⁸ CFU/mL). i, j Representative TEM images of ECN-pE and ECN-pE(C/A)₂ after exposure to SGF or 4% bile salt at 37 °C for 2 h from two independent samples. Scale bar: 1 μm. k Survival quantification of ECN-pE and ECN-pE(C/A)₂ exposed to 4% bile salt solution or simulated gastric fluid (SGF) for different times. l, m IVIS bioluminescence images of mice and their GI tract 3 h post-oral gavage by ECN-lux or ECN-lux(C/A)₂. ECN-lux ECN(pET28a-T5-luxCDABE). n Quantification evaluation of bioluminescence signals of ECN-lux and ECN-lux(C/A)₂ in mice. o–r Quantification analysis of living ECN-pE in the stomach, intestine, colon, and cecum 1, 3, 48, and 72 h post-oral gavage by ECN and ECN(C/A)₂. Data are presented as mean values ± SEM (n = 3 biologically independent samples for (a, b, e, f, k, and n), n = 6 biologically independent samples for (c and o–r)). Statistical analysis was evaluated with two-tailed Student’s t tests (*P  < 0.05, **P  < 0.01, ***P  <  0.001, ****P  < 0.0001). CFU colony-forming units, SGF simulated gastric fluid. Source data are provided as a Source Data file.

Fig. 3. Treatment efficacy of ECN-pE(C/A)2 against DSS-induced murine IBD.

a Schematic showing the experimental procedure for the treatment of DSS-induced IBD mice. C57BL/6 mice were given drinking water containing 3% DSS from day 0 to day 5. Meanwhile, the mice were fed PBS, ECN(C/A)_2, ECN-pE(C/A)₂ or ECN-pE (1 × 10⁸ CFU) on days 0, 2, 4, and 6 by gavage. b The body weight of the mice with different treatments. c The DAI of mice during the treatment. d Photographs and (e) corresponding quantified lengths of colons harvested from mice 10 days after different treatments. Scale bar: 2 cm. f The intestinal integrity function of mice was assessed by FITC-dextran assay after different treatments. g The colonic damage scores of mice after different treatments. h Representative images of H&E staining of colon tissue harvested on day 10 after different treatments from five biologically independent animals in each group. Scale bar: 200 μm. i The MPO activity in the colons of mice after different treatments. MPO, myeloperoxidase. j–n The levels of IL-1β, TNF-α, IL-6, IL-10, and TGF-β in the colon tissues measured by ELISA on day 10. Data are presented as mean values ± SEM (n = 5 biologically independent samples for (b, c, e–g, and i–n)). Statistical analysis was evaluated with two-tailed Student’s t tests (*P  < 0.05, **P  < 0.01, ***P  <  0.001, and ****P  <  00001). DSS dextran sodium sulfate. Source data are provided as a Source Data file.

Fig. 4. Reparative effect of ECN-pE(C/A)2 on the colonic epithelium and in vivo safety evaluation.

a, b Representative immunofluorescence staining images of colon sections to indicate the expression of tight junction proteins, including ZO-1 (a) and Occludin (b), 10 days after different treatments from two biologically independent animals. d, e Relative fluorescence intensity of colon sections as shown in (a) and (b). The nuclei were stained with DAPI (blue). Scale bar: 50 μm. c Representative TUNEL staining images of the colon tissues of mice in different groups from two biologically independent animals. f Relative fluorescence intensity of colon sections as shown in (c). The nuclei were stained with DAPI (blue). Scale bar: 50 μm. The mice were given PBS or ECN-pE(C/A)₂ (1 × 10⁸ CFU) on days 0, 2, 4, and 6 by gavage. g Complete blood and serum biochemistry data were obtained on day 10 after ECN-pE(C/A)₂ treatment. ALT alanine aminotransferase, AST aspartate aminotransferase, ALB albumin, BUN blood urea nitrogen. Data are presented as mean values ± SEM (n = 6 biologically independent samples for (d–f), n = 5 biologically independent samples for (g)). Statistical analysis was evaluated with two-tailed Student’s t tests (*P  < 0.05, **P  < 0.01, ***P  <  0.001, and ****P  <  0.0001). ZO-1 zonula occludens-1, DAPI 4’,6-diamidino-2-phenylindole, DSS dextran sodium sulfate. Source data are provided as a Source Data file.

Fig. 5. ECN-pE(C/A)2 modulates intestinal flora during IBD treatment.

a C57BL/6 mice were fed water containing several antibiotics (metronidazole, neomycin, vancomycin, and ampicillin) for 5 days and then switched to drinking water containing 3% DSS for 6 days. Then, the mice were treated with PBS, ECN(C/A)_2, ECN-pE(C/A)₂ or ECN-pE (1 × 10⁸ CFU) on days 0, 2, 4, and 6. b The body weight of mice during the treatment. c Photographs and (d) corresponding quantified lengths of colons harvested from mice 9 days after different treatments. Scale bar: 2 cm. e Representative H&E staining images of colon tissues harvested on day 9 after different treatments from five biologically independent animals in each group. Scale bar: 200 μm. f The DAI of mice during the treatment. g The MPO activity of colons after treatment. h Observed OTUs and (i) Shannon index of gut microbiota in mice after different treatments. OTUs operational taxonomic units. j Relative abundance of gut microbes at the phylum and family levels in mice. k Heatmap illustration of gut microbial distribution at the genus level. Relative abundance of Escherichia–Shigella (l), Lachnospiraceae_NK4A136 (m), and Odoribacter (n) collected from k. Data are presented as mean values ± SEM (n = 5 biologically independent samples for (b, d, f, and g), n = 7 biologically independent samples for (h–n)). Statistical analysis was evaluated with two-tailed Student’s t tests (*P  < 0.05, **P  < 0.01, ***P  <  0.001, and ****P  <  0.0001). DSS dextran sodium sulfate. Source data are provided as a Source Data file.

Fig. 6. Therapeutic efficacy of ECN-pE(C/A)2 against DSS-induced murine IBD with delayed treatment.

a Experimental procedure for the treatment of DSS-induced murine IBD. C57BL/6 mice were given drinking water containing 3% DSS from day 0 to day 5. Then, the mice were fed PBS, ECN(C/A)₂, ECN-pE(C/A)₂, or ECN-pE (1 × 10⁸ CFU) on days 5, 6, 7, and 9. b The body weight of mice during the treatment. c The DAI of mice during the treatment. d Photographs and (e) corresponding quantified lengths of colons harvested from mice after different treatments on day 10. Scale bar: 2 cm. f Representative H&E staining images of colon tissues harvested on day 10 after different treatments from five biologically independent animals in each group. Scale bar: 200 μm. g The MPO activity in the colon of mice after treatment. MPO myeloperoxidase. h–l The levels of IL-1β, TNF-α, IL-6, IL-10, and TGF-β in the colon tissues measured by ELISA on day 10 with delayed treatment. Data are presented as mean values ± SEM (n = 5 biologically independent samples for (b, c, e and g–l)). Statistical analysis was evaluated with two-tailed Student’s t tests (*P < 0.05, **P  < 0.01, ***P  <  0.001 and ****P<0.0001). DSS dextran sodium sulfate. Source data are provided as a Source Data file.