A T3SS Regulator Mutant HY9901 Delta araC of Vibrio alginolyticus Decreased the Expression of HopPmaJ and Provided Protection to Danio rerio as a Live-Attenuated Vaccine

Vibrio alginolyticus, a zoonotic bacterial pathogen, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the mutant HY9901 Delta araC was obtained from the laboratory and its biological characteristics were analyzed. The swimming ability of Delta araC decreased and exhibited a 2,600 times reduction in virulence to zebrafish. However, Delta araC showed no difference in growth and extracellular protease activity compared to wild type. Biofilm-forming ability was improved at 24 h, but no difference was observed at other time points. The results of drug sensitivity testing showed that compared with the wild-type HY9901 strain, Delta araC was sensitive to amikacin, tetracycline, neomycin, minocycline, and gentamicin. The transcription levels of T3SS effector proteins HopPmaJ, VopS, VcrV, and VopN were analyzed by qRT-PCR. The results showed that Delta araC had significantly upregulated the mRNA expression of VopS, VcrV, and VopN, but significantly downregulated the mRNA expression of HopPmaJ at each stage compared with HY9901. Western blotting and the beta-galactosidase reporter gene experiment also showed that the deletion of araC gene significantly downregulated the expression of HopPmaJ. Finally, the relative percent survival (RPS) rate of grouper inoculated by intramuscular (IM) injection of HY9901 Delta araC was 61.3% after being challenged with HY9901. Real-time qPCR analysis showed that vaccination of HY9901 Delta araC could enhance the expression of immune-related genes, including gata-1, il6, IgM, il-1 beta, and lyz in liver and spleen, indicating that Delta araC applied as a live-attenuated vaccine effectively induced an immune response in the zebrafish. This study provides evidence for the subsequent development of an effective live-attenuated V. alginolyticus vaccine.