The Role Of Important Functional Genes Of The Type Ⅵ Secretory System Of Pathogenic Aeromonas Hydrophila In Intracellular Survival

Aeromonas hydrophila,a common pathogen of human-zoo-fish,is one of the most common pathogens of fish,which can cause fulminant sepsis of fish and has brought huge economic losses to fishery production.In this study,the genome of A.hydrophila B11 strain was sequencd and analyzed.The virulence factors such as adhesion and invasion were predicted.The map of genes structure of VI secretory system of A.hydrophila B11 strain was drawn.There are 21 genes were found in the island that the genes of VI secretory system sequences assembled,including 11 structural genes and 10 regulatory protein genes.icmF and dotU in the upstream of the island and vgrG in the downstream.But hcp was not found in this island,which was quite diff-erent from other bacteria or even other strain of Aeromonas hydrophila.Then,RNA interference technology was used to stable silencing the expression of Hcp,VgrG,DotU and IcmF.And the silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi were constructed.The expression of these four silenced genes hcp,vgrG,dotU and icmF were detected by qPCR,and the results showed that the expression of these four genes was down-regulated by 77.1%,74.5%,70.8%and 94.42%,respectively.The intracellular survival experiment results of four silenced strains and B11 showed that the survival rate of wild type strains was 92.3%,and the survival rates of silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi were 7.69%,13.3%,13.5%and 20.58%,respectively,which were significantly lower than those of wild-type strain.The escape rate of wild-type strain was 20%,and the escape rate of silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi were 8.0%,9.4%,9.2%and 7.5%,respectively,which were significantly lower than that of wild-type strain.In order to further study the role and mechanism of Hcp,VgrG,DotU and IcmF in intracellular survival of A.hydrophila type VI secretory system,and to further compare the biological characteristics of silent and wild strains in terms of adhesion,biofilm formation,motility,anti-acid,anti-oxidation and hemolysis.The results of adhesion showed that the silenced strain Hcp-RNAi and VgrG-RNAi were 29%and 39%lower than that of the wild-type strain,respectively,which was significantly lower than that of wild-type strain,and the silenced strains DotU-RNAi and IcmF-RNAi were 48%and 52%lower than that of wild-type strain,extremely significantly lower than that of wild-type strain.The results of biofilm formation showed that compared with wild-type strain,the biofilm formation ability of silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi decreased by 11%,16%,13%and 8%,respectively,significantly lower than that of wild-type strain.The motility of silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi was decreased by 28.%,21%,27%and 59%,respectively,compared with that of the wild-type strain.In the bacterial hemolytic experiment,the hemolysis rate of wild-type strain was 86%,and the hemolysis rate of silenced strains Hcp-RNAi,VgrG-RNAi,DotU-RNAi and IcmF-RNAi were 71%,72%,72%,respectively,which was significantly lower than that of wild-type strain.The experiment of anti-acid and anti-oxidation showed that Hcp,VgrG;DotU and IcmF are not prominent in bacterial defense against ROS damage of fish macrophages,but play a certain role in bacterial against acid stress of fish macrophages.Baed on the above results,the following conclusions are made:(1)The genome composition and structure of the Ⅵ secretory system of bacteria are quite different in different bacteria or even different strains of the same species bacterium.(2)After the important genes(hcp,vgrG,dotU,icmF)of the type Ⅵ secretion system of A.hydrophila were silenced,the formation of T6SS was blocked.It can be speculated from the results of decline in movement,adhesion and biofilm that T6SS blocking of formation may affect the surface structure of bacterial cells,which in turn affects the intracellular survivala and escape of A.hydrophila.(3)Hcp,VgrG,DotU and IcmF can help bacteria survive and escape in macrophages by improving the acid resistance of bacteria,but their role in resisting ROS of host macrophages is not obvious.