Pathogenic “non-cholera” Vibrio species of Vibrio parahaemolyticus (V. parahaemolyticus) and Vibrio vulnificus (V. vulnificus) frequently pose a serious threat to aquaculture security and public health by causing infectious diseases. In this study, we reported the discovery of a marine-sourced antimicrobial peptide (AMP) called Ajapocin, which identified through a sequence optimization strategy. Ajapocin exhibited potent activity against V. parahaemolyticus and V. vulnificus pathogens, with minimum inhibitory concentrations (MICs) of 6–12 μM—comparable to the clinical agent Polymyxin B (PMB). In vivo, a single administration of Ajapocin (1 mg/mL) displayed therapeutic efficacy in a zebrafish-Vibrio infection model. Multiple doses reduced bacterial burden and accelerated wound healing in a mouse model of V. vulnificus-infected skin wounds. Ajapocin showed no cytotoxicity in ZF4 cells and HaCaT cells at concentrations up to 32 μM. Notably, after intraperitoneal injection for 1 week, Ajapocin did not induce cumulative hepatic or renal toxicity, as confirmed by histopathology analysis and chemistry profiles. Mechanistically, membrane-interacting Ajapocin targeted negative cellular components, enhancing membrane permeation, inducing membrane depolarization, and ultimately causing membrane damage and bacterial dysfunction. Taken together, these results position Ajapocin as an appealing anti-Vibrio agent for combating vibriosis in both aquaculture and clinical settings.
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