Identification And Functional Study Of Type ? Secreation System Novel Effector Protein In Aeromonas Hydrophila

Aeromonas hydrophila is a widely distributed gram-negative pathogen that has been recognised as a pathogen to human,livestock and fish.A.hydrophila can infect a wide range of host species,and the sporty aeromonas septicemia(MAS)caused by which is a serious hazard to freshwater aquaculture.A.hydrophila has a variety of virulence factors,the pathogenesis of it is complex.The type ? secreation system(T6SS)is an important virulence factor.Previously,the laboratory identified a set of T6SS encoded by A.hydrophila NJ-35.In this study,gene deletion technology,bacterial competition test and protein pull down assay were used to analyze the biological function of the putative T6SS effector protein gene in A.hydrophilia,to identify the effector protein and verify its function and secretion mechanism,to further elucidate the pathogenesis mechanism of T6SS.1 Biological functions of the type ? secretion system putative effector gene tle1AH1 and tle1AH2 in Aeromonas hydrophila NJ-35The aim is to identify the novel effectors of type ? secretion system(T6SS)and explore its function in Aeromonas hydrophila Chinese epidemic strain NJ-35,which is beneficial to further investigate the pathogenesis of pathogens.The putative effectors were screened in the whole genome of A.hydrophila NJ-35 based on the reported conservative domain DUF4123.Furthermore,the mutant and complementation strains were constructed by homologous recombination,and the competitiveness,growth,motility,biofilm formation,anti-phagocytosis and virulence in zebrafish were measured.Two putative genes was obtained and named tle1AH1(U876₁7550)and tle1AH2(U876₁7730),and their mutant(?tle1AH1 and ?tle1AH2)and complemented(C?tle1AHl and C?tle1AH2)strains were constructed.The mutant strains showed a significant decrease in anti-bacterial efficacy against Escherichia coli and Vibrio parahaemolyticus,but no effect on other Aeromonas of the same genus.And more than several times increase in LD50 over the wild-type strain.In addition,bio film formation ability of ?tle1AH1 increased significantly and phagocytic resistance of phagocytes was weakened.Biofilm formation ability of ?tle1AH2 decreased significantly.Meanwhile,the above characteristics of C?tle1AH1 and C?tle1AH2 were restored to the same level of the wide type strain.In this study,a functional analysis was conducted on two putative effector proteins of T6SS in A.hydrophila,which provides a basis for further research on the role of T6SS in the pathogenic mechanism of A.hydrophila.2 Validation of Aeromonas hydrophila NJ-35 type ? secretory system hypothesized effector-immunity pairIn order to verify the function and homologous immunity proteins of the putative effectors Tle1AH1 and Tle1AH2 in Aeromonas hydrophila,the toxicity mechanism of T6SS was further elucidated.A homologous recombination technique was used to construct effector-immunity gene deletion strains and corresponding homologous immunity genes complementary strains.The protective effect of immunity protein was verified by bacterial competition experiment.The results showed that no immune protection was observed for the complementary tli1AH1 in the mutant strain.In the tle1-tli1tli2AH2 mutant strain,complementary single tli1AH2 or tli2AH2 gene could not play an immune protection role,but complementary tli1tli2AH2 genes could play an immune protection role in the killing process of wild strain.Further,through the protein secretion test,it was found that Tle1AH2 was secreted by T6SS and had the phospholipase active motif GxSxG,and the core catalytic site was serine.The bacterial load in liver and pancreas,spleen and kidney was significantly higher than that of the missing strain after the wild strain and ?tle1AH2 strain co-infected crucian carp.The results above showed that Tle1-Tli1Tli2AH2 was an effector-immunity protein pair of T6SS in A.hydrophila,which played a role in bacterial colonization.3 Verification of the secreting mode of T6SS effector protein Tle1AH2 in A.hydrophila NJ-35Protein secretion have different ways of T6SS effectors,to verify whether the effector protein tle1AH2 of A.hydrophila interacts with other proteins during secretion,this chapter build Tle1AH2,Hcp and VgrG proteins of prokaryotic expression vector.Fusion protein expression was induced and purified,the purified protein for GST pull down test protein interactions.Results showed that the protein sizes were correct,validation of Western blot is ok,GST pull down showed that Tle1AH2 can interact with VgrG,but not be combined with Hcp,this indicated that the secretory process of Tle1AH2 depended on VgrG protein.In summary,this study analyzed the biological function of the putative effector protein genes,successfully identified the effector-immunity proteins Tlel-Tli1Tli2AH2 of T6SS in Aeromonas hydrophila,and proved that the effector protein is a kind of phospholipase and its specific secreted way through T6SS.It provides a theoretical basis for further elucidating the pathogenic mechanism of A.hydrophila.