A live attenuated strain of HY9901ΔvscB provides protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu)

Abstract

Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the T3SS chaperone protein gene vscB was cloned from V. alginolyticus wild-type strain HY9901and the vscB gene has a length of 429 bp and encodes 142 amino acids. The amino acid sequence homology comparison results showed that VscB had the highest similarity with that of Vibrio parahaemolyticus VscB, reaching 91%. The mutant strain HY9901ΔvscB was constructed by the in-frame deletion method. The HY9901ΔvscB showed an attenuated swarming phenotype and a 23-fold decrease in virulence to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu). However, the HY9901ΔvscB showed no difference in morphology, growth, biofilm formation and extracellular protease (ECPase) activity. The results of antibiotic susceptibility testing showed that the wild-type HY9901 is more sensitive to Doxycycline, Minocyline and Kanamycin than in the HY9901ΔvscB. A total of 95 differentially expressed genes were screened by transcriptome sequencing analysis of HY9901 and strain ΔvscB, revealing 57 genes up-regulated and 38 genes were down-regulated in the ΔvscB compared to HY9901. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes showing that ΔvscB had decreased expression of vopN and vscO and increased expression of hop, vscN, vscK, vscL and vopS compared to HY9901. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901ΔvscB had a relative percent survival (RPS) of 77.6% following challenge with HY9901. Real-time qPCR assays showed that vaccination with HY9901ΔvscB enhanced the expression of immune-related genes, including MHC-I, IgM, and CD8α both in the liver and spleen, indicating that HY9901ΔvscB is able to induce humoral and cell-mediated immune responses in pearl gentian grouper. These results demonstrate that the HY9901ΔvscB mutant could be used as an effective live attenuated vaccine to combat V. alginolyticus infection in pearl gentian grouper.