{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T16:42:08Z","timestamp":1791218528769,"version":"4.1.0"},"reference-count":66,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T00:00:00Z","timestamp":1782950400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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-\u03b1) 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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We engineered\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      Nissle 1917 (EcN) to secrete the anti-TNF-\u03b1 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\u2212murine TNF-\u03b1 (mTNF-\u03b1) 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Among the tested signal peptides, \u03b1-hemolysin (HlyA) achieved highest secretion (4.6\u00a0mg\/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 (EC\n                      <jats:sub>50<\/jats:sub>\n                      27.9\u00a0nM) and potent suppression of LPS-induced mRNA expression of\n                      <jats:italic>Tnf<\/jats:italic>\n                      and interleukin-1\u03b2 (\n                      <jats:italic>Il1b<\/jats:italic>\n                      ) 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\n                      <jats:italic>Tnf<\/jats:italic>\n                      and\n                      <jats:italic>Il1b<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This study establishes EcN-MT1 as a potent, orally deliverable live biotherapeutic that achieves localized TNF-\u03b1 neutralization while concurrently promoting microbial and mucosal homeostasis, offering a novel and translatable strategy for IBD treatment.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1865862","type":"journal-article","created":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T04:23:41Z","timestamp":1782966221000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Engineered Escherichia coli Nissle 1917 secreting anti-TNF-\u03b1 nanobody as a single-strain live biotherapeutic for inflammatory bowel disease"],"prefix":"10.3389","volume":"17","author":[{"given":"Qiumei","family":"Zhu","sequence":"first","affiliation":[{"name":"School of Medicine and Pharmacy, Ocean University of China","place":["Qingdao, China"]},{"name":"Qingdao Center of Technology Innovation for Shark Antibody Development, College of Biological Engineering, Qingdao University of Science and Technology","place":["Qingdao, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shitao","family":"Feng","sequence":"additional","affiliation":[{"name":"School of Medicine and Pharmacy, Ocean University of China","place":["Qingdao, China"]},{"name":"Qingdao Center of Technology Innovation for Shark Antibody Development, College of Biological Engineering, Qingdao University of Science and Technology","place":["Qingdao, 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of Science and Technology","place":["Qingdao, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changning","family":"Sun","sequence":"additional","affiliation":[{"name":"Qingdao Center of Technology Innovation for Shark Antibody Development, College of Biological Engineering, Qingdao University of Science and Technology","place":["Qingdao, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingqiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Qingdao Restore Medical Laboratory Co., Ltd.","place":["Qingdao, Shandong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinrui","family":"Li","sequence":"additional","affiliation":[{"name":"Qingdao Center of Technology Innovation for Shark Antibody Development, College of Biological Engineering, Qingdao University of Science and Technology","place":["Qingdao, 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