Engineered Antimicrobial Peptide Z-d14CFR Protects Against Mastitis through Modulation of Drp1-Mediated Host Mitochondrial Dynamics.

Bovine mastitis caused by enteropathogenic Escherichia coli (EPEC) poses a significant threat to the dairy industry, and antimicrobial resistance underscores the urgent need for novel therapeutics. Here, we show that the engineered antimicrobial peptide Z-d14CFR exerts dual protective effects against EPEC-induced mastitis through direct bactericidal activity and host-directed modulation of mitochondrial dynamics. Z-d14CFR disrupts bacterial membranes, induces oxidative stress in EPEC, and alleviates mammary tissue damage and NLRP3 inflammasome activation in a murine mastitis model. Mechanistically, EPEC infection induces dynamin-related protein 1 (Drp1)-mediated mitochondrial hyper-fission and dysfunction, whereas Z-d14CFR restores mitochondrial dynamics by promoting fusion protein expression and suppressing excessive fission, thereby reducing ROS production and downstream NLRP3-driven inflammation. Using pharmacological inhibition and genetic modification (knockout and overexpression) of Drp1, we found that the protective effects of Z-d14CFR are dependent on Drp1-mediated mitochondrial dynamics. These findings indicate Z-d14CFR as a promising dual-function candidate for combating bacterial mastitis and addressing antimicrobial resistance.