Engineering of copper-binding probiotic for the treatment of copper poisoning in a drinking water-induced mouse model

Copper poisoning is a serious condition marked by multi‑organ toxicity, yet existing treatments suffer from limited efficacy and adverse effects, necessitating safer and more effective copper‑depletion approaches. In this study, we aimed to engineer a probiotic strain capable of sequestering intestinal copper and evaluate its therapeutic potential in a mouse model of copper poisoning. We engineered Escherichia coli Nissle 1917 to surface‑display various copper‑binding elements and identified the strain overexpressing Tff1 (EcN‑Tff1) as exhibiting the highest copper‑binding capacity. Oral administration of EcN‑Tff1 in a mouse model of copper poisoning, established by copper‑supplemented drinking water, significantly alleviated disease phenotypes, as evidenced by improved body weight, reduced hepatic inflammation, and attenuated liver pathology. EcN‑Tff1 treatment increased fecal copper excretion while decreasing copper accumulation in the liver and urine, indicating enhanced copper elimination via the intestinal route. 16S rRNA amplicon sequencing revealed that EcN‑Tff1 administration altered gut microbiota composition, including changes in the relative abundances of Ligilactobacillus , Eubacterium siraeum group , Coriobacteriaceae_UCG‑002 , and Faecalibaculum . Functional prediction using PICRUSt2 showed that the engineered strain modulated gut microbial metabolic pathways, with enrichment in pathways related to signal transduction, cofactor and vitamin metabolism, xenobiotic biodegradation, amino acid metabolism, and pathogen infection. Our study demonstrates that EcN‑Tff1 serves as an effective oral bioagent for reducing copper burden and provides a promising microbe‑based therapeutic strategy for copper overload disorders.