Study on biological characteristics and pathogenicity of VPA1500 gene knock-out mutant of Vibrio parahaemolyticus

Abstract:

The type Ⅵ secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Vibrio parahaemolyticus also harbours T6SS gene clusters and possesses two T6SSs, T6SS1 and T6SS2. [Objective] In previous work, we identified several potential T6SS effectors by using comparative proteomics. In this study, VPA1500 was chosen to explore the roles on biological characteristics and pathogenicity of Vibrio parahaemolyticus.[Methods] The deletion mutant ΔVPA1500 and complementary strain CΔVPA1500 were constructed by using homologous recombination technology. Growth characteristics, anti-bacterial activity in vivo, motility, the transcription level of flagella-related genes, and biofilm formation ability were analyzed in the wild-type strain (WT), ΔVPA1500 and CΔVPA1500. Furthermore, cytotoxicity to host cell, lethality rate in mice, bacterial colonization, and histopathological changes were also analyzed between WT and ΔVPA1500. [Results] Compared with WT, there were no significant changes in growth characteristics, swarming ability, and biofilm formation of ΔVPA1500 (P>0.05), while swimming ability was significantly decreased (P<0.05). Transmission electron micrographs showed that VPA1500 deletion affected the formation of bacterial flagella in V. parahaemolyticus. qPCR results also showed that the VPA1500 gene significantly inhibited the transcription level of some flagella-related genes in WT. Bacterial competition experiments showed that the deletion of VPA1500 reduced the anti-bacterial activity of Vibrio parahaemolyticus against E. coli in vitro. However, ΔVPA1500 showed significantly weaker cytotoxicity to Hela cells than WT. In addition, ΔVPA1500 exhibited attenuated virulence in mice that showed lower a lethality rate than that of the wild-type strain. Moreover, VPA1500 deletion affected the colonization in heart, liver, spleen, and kidney of mice, whereas the complementation strain restored the virulence to resemble that of WT. Histopathological analyses further demonstrated that detection of VPA1500 could reduce the damage of Vibrio parahaemolyticus to tissues in mice. [Conclusion] The VPA1500 plays an important role in swimming motility, pathogenicity, and anti-bacterial activity in V. parahaemolyticus.