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

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

JOURNAL=Frontiers in Veterinary Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.938822

DOI=10.3389/fvets.2022.938822

ISSN=2297-1769

ABSTRACT=<p><italic>Vibrio alginolyticus</italic>, 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 <italic>V. alginolyticus</italic>. In this study, the T3SS regulatory gene <italic>exsA</italic>, which was cloned from <italic>V. alginolyticus</italic> wild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The Δ<italic>exsA</italic> was constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and Δ<italic>exsA</italic>, the Δ<italic>exsA</italic> exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the Δ<italic>exsA</italic> showed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901Δ<italic>exsA</italic> 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<italic>exsA</italic>, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels of <italic>hop</italic> gene revealing that <italic>exsA</italic> gene could facilitate the expression of <italic>hop</italic> gene. Finally, <italic>Danio rerio</italic>, vaccinated with Δ<italic>exsA</italic> 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<italic>exsA</italic> increased the expression of immune-related genes, including <italic>GATA-1, IL6, IgM</italic>, and <italic>TNF-</italic>α in zebrafish. In summary, these results demonstrate the importance of <italic>exsA</italic> in <italic>V. alginolyticus</italic> and provide a basis for further investigations into the virulence and infection mechanism.</p>