Functional characterization of T3SS C-ring component VscQ and evaluation of its mutant as a live attenuated vaccine in zebrafish (Danio rerio) model

Abstract

Vibrio alginolyticus, a Gram-negative bacterium, has been recognized as an opportunistic pathogen in marine animals as well as humans. Type III secretion system (T3SS) is critical for pathogen virulence and disease development. However, no more information is known about the C-ring component VscQ and its physiological role. In this study, gene vscQ was cloned from V. alginolyticus wild-type strain HY9901 and the mutant strain HY9901ΔvscQ was constructed by the in-frame deletion method. The HY9901ΔvscQ mutant showed an attenuated swarming phenotype and a closely 4.6-fold decrease in the virulence to Danio rerio. However, the HY9901ΔvscQ mutant showed no difference in growth, biofilm formation and ECPase activity. HY9901ΔvscQ reduces the release of LDH, NO and caspase-3 activity of infected FHM cell, which are involved in fish cell apoptosis. Deletion of gene vscQ downregulates the expression level of T3SS-related genes including vscL, vopB, hop, vscO, vscK, vopD, vcrV and vopS and flagellum-related genes (flaA and fliG). And Danio rerio vaccinated via i.m injection with HY9901ΔvscQ induced a relative percent survival (RPS) value of 71% after challenging with the wild-type HY9901. Real-time PCR assays showed that vaccination with HY9901ΔvscQ enhanced the expression of immune-related genes, including TNF-α, TLR5, IL-6R, IgM and c/ebpβ in liver and spleen after vaccination, indicating that it is able to induce humoral and cell-mediated immune response in zebrafish. These results demonstrate that the HY9901ΔvscQ mutant could be used as an effective live vaccine to combat V. alginolyticus infection.