Evaluation of safety and efficacy of an auxotrophic aroA mutant live attenuated vaccine against piscine Streptococcus agalactiae infection

Streptococcus agalactiae is a major pathogen threatening global tilapia aquaculture, causing severe economic losses due to high mortality. The rise of antimicrobial resistance necessitates the development of effective vaccines for streptococcosis control. Here, we generated an auxotrophic mutant through the targeted deletion of the aroA gene, which encodes a key enzyme in aromatic amino acid biosynthesis. The aroA mutant (ΔaroA) exhibited reduced intracellular survival within macrophages, a phenotype that was not restored by supplementation with exogenous aromatic amino acids under our experimental conditions. These findings suggest that aroA contributes to intracellular survival through mechanisms extending beyond its role in aromatic amino acid biosynthesis. In zebrafish, Nile tilapia and mouse models, ΔaroA demonstrated stable attenuation, reduced endothelial cell damage, and mitigated blood-brain barrier disruption and neuroinflammation, confirming its safety. The ΔaroA strain provided dose-dependent protection against lethal S. agalactiae challenges in both tilapia and mice, with 100% protection in tilapia conferred at a dose of 10⁶ CFU following intraperitoneal administration. This study represents the first investigation into the role of aroA in S. agalactiae pathogenicity, providing valuable insights into the mechanisms of micronutrient utilization during bacterial pathogenesis. Also, our findings strongly support auxotrophic mutation as a promising attenuation strategy for vaccine development against streptococcosis in aquatic species.