Study Of Potential T6SS1 Effector In Vibro Parahaemolyticus

Vibrio parahaemolyticus,a gram-negative bacterium,is a common food-borne pathogen that is mainly distributed in a variety of seafood and causes huge economic losses to the aquaculture industry.Secreted toxins are important arms used by bacteria to adjust their metabolic state to colonize a niche in a hostile environment.The study of virulence factors has become a new method to understand the pathogenic mechanism of Vibrio parahaemolyticus.Bacteria have evolved several discrete pathways to recognize their effector proteins with extraordinary fidelity and translocate them to their surroundings,into the host cells or rival bacterial cells.Type Ⅵ secretion system(T6SS),bearing functional homology to an inverted T4-phage contractile tail,is one such pathway deployed by many gram-negative proteobacteria.In this study,we selected two potential effectors to prove their effects on the biological characteristics and pathogenicity of Vibrio parahaemolyticus by constructing corresponding deletion strains and complementary strains through homologous recombination technology.Experiment 1 Construction of VPA1500 and VP2659 gene knock-out mutants of type Ⅵsecretion system 1 effector of Vibrio parahaemolyticusGram-negative bacteria use the secretion system to transport effectors from the cytoplasm to the extracellular environment,to interact with the surrounding environment or microenvironment.Type Ⅵ secretion system(T6SS)is a virulence factor secretion system of bacteria,which participates in regulating the environmental adaptability and virulence through secreted proteins.We constructed Δvip A1-hcp1 of Vibrio parahaemolyticus in the early stage,compared and analyzed the secreted differential protein of SH112 and Δvip A1-hcp1 through differential proteomics technology(lab-free Quantitation).In this experiment,we selected an unknown protein VPA1500 and a BolA/Yrb A family protein VP2659 and constructed deletion strains and complementary strains respectively.Genetic stability analysis by PCR showed that two gene-deleted strains and complementary strains could be inherited stably.Compared with WT,there were no significant changes in growth characteristics between ΔVPA1500 and ΔVP2659.Experiment 2 Study on biological characteristics and pathogenicity of VPA1500 gene knock-out mutant of Vibrio parahaemolyticusIn this study,VPA1500 was chosen to explore the roles on biological characteristics and pathogenicity of Vibrio parahaemolyticus.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.The results showed that there were no significant changes in swarming ability,and biofilm formation of ΔVPA1500 and WT while swimming ability was significantly decreased.Transmission electron micrographs showed that VPA1500 deletion affected the formation of bacterial flagella in V.parahaemolyticus.q PCR 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.Besides,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 the 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.All of the above results showed that the VPA1500 plays an important role in swimming motility,pathogenicity,and anti-bacterial activity in V.parahaemolyticus.Experiment 3 The effect of BolA family protein VP2659 on the biological characteristics and pathogenicity of Vibrio parahaemolyticusIn this study,we identified a novel BolA transcription factor,VP2659,which influences the biological characteristics and pathogenicity of Vibrio parahaemolyticus.The experimental results showed that compared with WT and CΔVP2659,the VP2659 deletion mutant(ΔVP2659)eliminated the swarming motility and biofilm formation ability.Interestingly,the swimming ability was increased.The RT-PCR results further showed that the deletion of the VP2659 gene would up-regulate the transcription level of polar flagellarelated genes of Vibrio parahaemolyticus.ΔVP2659 not only reduced cytotoxicity to Hela cells and mouse virulence but also reduced the colonization ability in mice.Thus,this study illustrates BolA transcriptional regulatory factors can affect the biological characteristics and pathogenic process of Vibrio parahaemolyticus.