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

, 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 . In this study, the T3SS regulatory gene , which was cloned from wild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The Δ was constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and Δ, the Δ exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the Δ showed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901Δ 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 Δ, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels of gene revealing that gene could facilitate the expression of gene. Finally, , vaccinated with Δ 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 Δ increased the expression of immune-related genes, including , and α in zebrafish. In summary, these results demonstrate the importance of in and provide a basis for further investigations into the virulence and infection mechanism.