Analysis And Functional Study Of The Extracellular Protein And Hisj Gene Of Aeromonas Veronii

Aeromonas veronii is a kind of human-animal-aquatic pathogenic bacteria with a wide range of pathogenicity.The infected fish with Aeromonas veronii often had ulcers and septicemia even causing death.The infected human with Aeromonas veronii will have symptoms such as diarrhea and gastroenteritis.Extracellular products?Extracellular products,ECPs?are bacteria metabolites released to the environment during the process of growth and reproduction;they can also invade the host organism,diffuse in the host reproduction,and resistance to host immune factors,causing the host disease,and even death.The extracellular products mainly include extracellular enzymes,such as toxins and enterotoxin etc,which play an important role for pathogen virulence.The reports about Aeromonas veronii are increasing in recent years,which focus on the aspect of pathogenic ability,but few research on its pathogenesis.At present,domestic and international studies have found that extracellular products from most Aeromonas veronii can cause aquatic animals death,but the protein in the extracellular product is unknown to play a pathogenic role.Therefore,research on the relationship between protein and pathogenicity in Aeromonas veronii extracellular products will provide a basis for further understanding of the pathogenesis of Aeromonas veronii.In this study,the extracellular product of Aeromonas veronii TH0426 was successfully extracted,and the protein components of extracellular products were determined by the combination of LC-MSMS.The results showed that the Extracellular product of Aeromonas veronii TH0426?Pelteobagrus fulvidraco source?contained 167 kinds of proteins.In addition,we used the same method to extract and determine the rest of the five kinds of different source.AV161?snakehead source?extracellular product contained 118 kinds of proteins;CVCC3700?fox source?extracellular product contained 395 proteins;ATCC35624?human source?extracellular product contained 189 proteins;QXF0711B?prawns source?extracellular product contained 90 proteins;CC-8155?grass carp source?extracellular product contained 197 proteins.Moreover,the extracellular products from the five different sources were compared with that of the Aeromonas veronii TH0426.The shared protein by the six bacteria was binding protein,encoding hisJ gene.The differentially expressed proteins of TH0426 extracellular product by GO analysis showed that secreted protein can participate in 872 biological processes.One hundred and fifty of them with significant difference?P<0.05?,mainly involved in amino acid decomposition and biological synthesis process,Participate in 454 molecular functions.Ninety of them with significant difference?P<0.05?mainly involved in transporters,catalytic proteins and periplasmic binding proteins.In order to explore the function of hisJ gene,the deletion mutants?hisJ was successfully constructed using suicide plasmid pRE112 through homologous recombination method,and the complementary strains C-hisJ?pACYC?and C-hisJ?pBBR?using expression plasmid pBBR1-MCS and pACYC-184.PCR results showed that all the three strains were stable for more than 40 generations.Moreover,the qRT-PCR results showed that the expression of hisJ gene in the complementary strain C-hisJ?pBBR?was significantly higher than that of the complementary strain C-hisJ?pACYC?.Therefore,we chose the complementary strain C-hisJ?pBBR?for the analysis of biological characteristics and subsequent experiments.The results of biological characteristics showed that bacterial growth morphology,hemolysis activity and swimming ability have no obvious changes,but the swimming and biofilm formation ability significantly diminished deleting hisJ gene of Aeromonas veronii TH0426.Using the flagella staining and transmission electron microscope,we also found that flagella fell off easily after missing hisJ gene.In addition,compared with the wild strain,the adhesive capacity of the missing strains decreased by 10 times,and the virulence of EPC cells decreased significantly.The pathogenic test in zebrafish and mice indicated that the LD₅₀ of missing strain in zebrafish increased 865 times and virulence declined,which showed extremely significant difference?p<0.001?.The LD₅₀ in mice has increased 2.8 times and virulence declined,showing that the pathogenicity of the missing strain in zebrafish and mice were significantly decreased.Compared with wild strains TH0426 group,the content of tissue bacteria in carp toxicity attack test decreased significantly in heart,liver,spleen and kidney,and pathogenic strength was decreased obviously.In conclusion,the variety of proteins in six different sources of Aeromonas veronii extracellular product was analyzed by LC-MSMS.Compared with the Aeromonas veronii extracellular,the shared protein by the six bacteria extracellular was binding protein,encoding hisJ gene.The biological characteristics of the missing and complementary strains of Aeromonas veronii TH0426 were analyzed.After missing hisJ gene,the flagella of Aeromonas veronii TH0426 is easy to fall off,and the biofilm formation ability and the animal's pathogenic ability are obviously weakened.The results of this study provide a basis for elucidating the pathogenesis of Aeromonas veronii.