AUTHOR=Wang Junlin , Zeng Fuyuan , Feng Haiyun , Chen Liangchuan , Zhang Weijie , Xiao Xing , Monaghan Sean J. , Jian Jichang , Pang Huanying , Wang Na 

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

JOURNAL=Frontiers in Marine Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/marine-science/articles/10.3389/fmars.2022.938742

DOI=10.3389/fmars.2022.938742

ISSN=2296-7745

ABSTRACT=<p><italic>Vibrio alginolyticus</italic>, 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 Δ<italic>araC</italic> was obtained from the laboratory and its biological characteristics were analyzed. The swimming ability of Δ<italic>araC</italic> decreased and exhibited a 2,600 times reduction in virulence to zebrafish. However, Δ<italic>araC</italic> 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, Δ<italic>araC</italic> 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 Δ<italic>araC</italic> 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 β-galactosidase reporter gene experiment also showed that the deletion of <italic>araC</italic> gene significantly downregulated the expression of HopPmaJ. Finally, the relative percent survival (RPS) rate of grouper inoculated by intramuscular (IM) injection of HY9901 Δ<italic>araC</italic> was 61.3% after being challenged with HY9901. Real-time qPCR analysis showed that vaccination of HY9901 Δ<italic>araC</italic> could enhance the expression of immune-related genes, including <italic>gata-1</italic>, <italic>il6</italic>, <italic>IgM</italic>, <italic>il-1β</italic>, and <italic>lyz</italic> in liver and spleen, indicating that Δ<italic>araC</italic> 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 <italic>V. alginolyticus</italic> vaccine.</p>