An engineered probiotic Escherichia coli Nissle 1917 strain for Amuc_1100 delivery in a CT26 colorectal tumor model.

Engineered probiotics provide a microbial platform for the localized delivery of bioactive proteins. Escherichia coli Nissle 1917 (EcN) is an attractive bacterial chassis for this purpose because of its ability to colonize tumors and its genetic tractability. Amuc_1100, an outer membrane protein of Akkermansia muciniphila, has shown antitumor and immunomodulatory effects after oral administration. However, whether EcN-based delivery can improve the antitumor performance of microbiome-derived proteins such as Amuc_1100 remains unclear. Here, EcN was engineered with a pelB-His-Amuc_1100 expression cassette to generate EcN(pAmuc), which was evaluated in an immunocompetent CT26 colorectal tumor model. After a single intravenous administration, viable EcN(pAmuc) remained recoverable from tumors for up to 21 days, and His-tagged cargo-related protein signals were detected in tumor tissue at the study endpoint. EcN(pAmuc) reduced tumor growth and prolonged survival compared with untreated CT26-bearing mice and the oral recombinant Amuc_1100 regimen, with median survival increasing from 21 to 33 days relative to the untreated group. Treatment was accompanied by an increase in intratumoral CD3-positive cell proportion from 3.39% to 9.35% and TUNEL positivity from 5.08% to 10.41%, together with lower Ki-67 positivity and intratumoral TGF-β levels and a higher splenic CD8⁺ T-cell proportion. Short-term safety-related assessments, including body weight, organ weights, peripheral white blood cell counts, serum creatinine, and major-organ histology, revealed no clear treatment-associated abnormalities under the conditions tested. These findings support EcN as a delivery chassis for Amuc_1100 and provide a basis for further optimization of this engineered probiotic platform in colorectal tumor models.