Calvatia lilacina spores extract and engineered E. coli Nissle 1917 remodel the diabetic wound microenvironment through combined treatment.

Diabetic wound healing is severely impaired by persistent inflammation and microbial dysbiosis. The study presents a combinedfunctional ingredient system centered on an engineered thymidine-auxotrophic Escherichia coli Nissle 1917 strain (ThyA-/--EcN), whose survival and activity are sustained by the thymidine naturally present in Calvatia lilacina spore extract (CLSE). CLSE was chemically characterized by UHPLC-QTOF-MS, and its thymidine content was quantified as 4.1 mg/g by HPLC. In a full-thickness excisional wound model in diabetic db/db mice, CLSE alone accelerated wound healing, enhanced re-epithelialization and collagen deposition, and restructured the wound microbiota by increasing Escherichia-Shigella while suppressing Staphylococcus (SA). Notably, the combination of CLSE and ThyA-/--EcN significantly prolonged the colonization of the engineered probiotic in vivo compared to ThyA-/--EcN alone, an effect we hypothesize to be attributable, at least in part, to the thymidine provided by CLSE. This prolonged colonization, in combination with CLSE, boosted M2 macrophage polarization, enhanced fibroblast function, and strengthened the suppression of SA, which our in vitro data suggest may involve nutrient competition. The results demonstrate that CLSE not only directly improves wound healing but also acts as a natural thymidine delivery vehicle to unlock the full potential of ThyA-/--EcN, establishing a rationale for developing a food-derived bioactive extract-assisted live biotherapeutic strategy.