Abstract

Vibrio anguillarumis a major pathogenic bacterium causing vibriosis in aquatic animals, leading to substantial economic losses in the global aquaculture industry. Previous studies have indicated that L-arginine modulates the virulence of the pathogen, but the underlying molecular mechanisms remain elusive. The present study aimed to clarify the regulatory role of L-arginine metabolism inV. anguillarumvirulence. We first evaluated the effects of L-arginine and its major metabolites (agmatine, putrescine, spermine) on the hemolytic activity ofV. anguillarum. Results showed that L-arginine and its metabolites regulated hemolytic activity in a concentration-dependent biphasic manner, with agmatine exerting the most potent promoting effect. To identify the critical metabolic branch involved, four isogenic mutants were constructed targeting key genes in arginine metabolism (adc,astA,astD). Phenotypic analysis revealed that only theadcdeletion mutant (Δadc) exhibited near-complete loss of hemolytic activity, which was dose-dependently restored by supplementation with agmatine, putrescine, or spermine. Transcriptomic analysis identified 704 significantly differentially expressed genes (DEGs) between Δadcand WT strains, with downregulated DEGs enriched in virulence-associated pathways. Key hemolysin and secretion system genes were validated to be downregulated in ∆adcby quantitative real-time PCR (qRT-PCR). Additionally, Δadcdisplayed attenuated anti-phagocytic ability in Tetrahymena co-culture assays, impaired biofilm formation, and increased susceptibility to multiple classes of antibiotics. Collectively, our findings demonstrate that L-arginine modulatesV. anguillarumhemolysis and overall virulence through theadc-mediated agmatine biosynthesis branch. This study fills the knowledge gap in the regulatory mechanism of L-arginine onV. anguillarumvirulence and provides a potent