Engineered Lactococcus lactis expressing antimicrobial peptide HI: Enhanced survival and protection against ETEC in mice.

The rising prevalence of antibiotic resistance underscores the urgent need for alternative strategies to manage pathogenic bacteria. Engineered probiotics offer a promising platform for delivering antimicrobial peptides (AMPs); however, their practical application remains constrained by challenges related to maintaining viability and in vivo functionality. This study focused on two main aspects: (1) optimizing a freeze-drying strategy for Lactococcus lactis/pNZC-Usp45-H-6 ×His (L. L/HI), which expresses the AMP HI targeting Escherichia coli, and (2) evaluating its protective efficacy against enterotoxigenic Escherichia coli (ETEC) infection in a murine model. Sorbitol at a concentration of 6 % (w/v) was identified as the most effective cryoprotectant for preserving bacterial viability after freeze-drying. In the ETEC infection model, oral administration of L. L/HI significantly alleviated intestinal injury by reducing bacterial colonization and lipopolysaccharide levels, alleviating inflammation, and restoring the expression of tight junction genes. Moreover, L. L/HI downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and upregulated the anti-inflammatory cytokine IL-10 in ileal tissues. These findings demonstrate that oral administration of L. L/HI reduced the bacterial burden in the ileum of ETEC-infected mice and indirectly alleviated inflammation and intestinal barrier damage caused by ETEC infection. This study provides a novel approach for the translational application of engineered probiotics.