Functional characterization of Vibrio alginolyticus T3SS regulator ExsA and evaluation of its mutant as a live attenuated vaccine candidate in zebrafish (Danio rerio) model

Weijie Zhang^{1, 2}, Liangchuan Chen^{1, 2}, Haiyun Feng^{1, 2}, Junlin Wang^{1, 2}, Fuyuan Zeng^{1, 2}, Xing Xiao^{1, 2}, Jichang Jian^{1, 2}, Na Wang³ and Huanying Pang^{1, 2}

• ¹Fisheries College, Guangdong Ocean University, China
• ²Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, China
• ³Chinese Academy of Inspection and Quarantine, China

Vibrio alginolyticus, a Gram-negative bacterium, is an opportunistic pathogen of both marine animals and humans, resulting in significant losses in the aquaculture industry. Type III secretion system (T3SS) is a crucial virulence mechanism of V. alginolyticus. In this study, the T3SS regulatory gene exsA, which was cloned from V. alginolyticus wild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The ΔexsA was constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and ΔexsA, the ΔexsA exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the ΔexsA showed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901ΔexsA was more sensitive to kanamycin, minocycline, tetracycline, gentamicin, doxycycline and neomycin. Compared to HY9901 there were 541 up-regulated genes and 663 down-regulated genes in ΔexsA, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels of hop gene revealing that exsA gene could facilitate the expression of hop gene. Finally, Danio rerio, vaccinated with ΔexsA through intramuscular injection, induced a relative percent survival (RPS) value of 66.7% after challenging with HY9901 wild type strain. qRT-PCR assays showed that vaccination with ΔexsA increased the expression of immune-related genes, including GATA-1, IL6, IgM, and TNF-α in zebrafish. In summary, these results demonstrate the importance of exsA in V. alginolyticus and provide a basis for further investigations into the virulence and infection mechanism.