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

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 Delta exsA was constructed by homologous recombination and Overlap-PCR. Although there was no di erence in growth between HY9901 and Delta exsA, the Delta exsA exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the Delta exsA showed a weakened swarming phenotype and an similar to 100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901 Delta exsA wasmore sensitive to kanamycin, minocycline, tetracycline, gentamicin, doxycycline and neomycin. Compared to HY9901 there were 541 up-regulated genes and 663 down-regulated genes in Delta exsA, screened by transcriptome sequencing. qRT-PCR and beta-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 Delta exsA 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 Delta exsA increased the expression of immune-related genes, including GATA-1, IL6, IgM, and TNF-alpha 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.