The Role And Mechanism Of Two-Component System EnvZ/OmpR In The Pathogenicity And Intracellular Survival Of Aeromonas Hydrophila

Aeromonas hydrophila is a pathogenic bacterium widely distributed in the world and can cause human-animal-fish co-occurrence.It is one of the most common pathogenic bacteria in aquaculture,which can cause explosive sepsis in fish and bring huge economic losses to the aquaculture industry.Our previous research suggested that the pathogenic A.hydrophila B11 strain can survive in fish macrophages,indicating that A.hydrophila has a mechanism to resist macrophage damage.Bacterial survival in host macrophages may be one of the reasons for the difficulty in treating diseases and the emergence of drug-resistant strains.The two-component system EnvZ/OmpR plays an important regulatory role in Gram-negative bacteria,and participates in the regulation of bacterial pathogenicity,virulence,intracellular survival,biofilm formation and other physiological functions.In this study,the A.hydrophila B11 strain was used as the research object,using RNAi technology and comparative transcriptomics to analyze and study the EnvZ/OmpR two-component system in the pathogenicity and intracellular survival of A.hydrophila Mechanism.First,the A.hydrophila B11 silent strains envZ-RNAi and ompR-RNAi were constructed,and the comparative transcriptome analysis was performed on the wild strain and the silent strain.The transcriptome analysis results showed that:The expression of hlyA,hns,ast,ffh,gspH,gspG,motA,che V,cheY,fliD,htpG,clpB,mcp,exbB,glpA,glpB,glpD,trpE and other genes has changed significantly after the two-component system EnvZ/OmpR was silenced.According to the literature,the functions of these genes are related to the type Ⅱ secretion system,movement,chemotaxis,biofilm formation and adhesion of bacteria.Therefore,the expression and function of these genes were further verified.The results showed that compared with the wild-type strain B11,the chemotaxis ability of silent strains envZ-RNAi and ompR-RNAi decreased by 44.5%and 45.5%,the adhesion ability decreased by 9.5%and 14.9%,and biofilm formation ability decreased by 7.6%and 15.1%.In addition,the artificial infection test results showed that the LD50 of the B11,envZ-RNAi and ompR-RNAi was 2.98 ×104 cfu/ml,2.11×105 cfu/ml,and 2.21 ×105 cfu/ml,respectively.Overall,the LD50 of the silent strain is one order of magnitude lower than that of the wild strain.Based on the above results,it can be speculated that the two-component system EnvZ/OmpR can affect the pathogenicity of A.hydrophila by regulating the type Ⅱ secretion system,chemotaxis,adhesion and biofilm formation of this bacterium.Since the intracellular survival ability of bacteria is also one of the virulence factors,this study further explores the role and mechanism of the two-component system EnvZ/OmpR in the intracellular survival of A.hydrophila.The results showed that compared with the wild strain the intracellular survival ability of the silent strains envZ-RNAi and ompR-RNAi decreased by 91.1%and 83.7%.Transcriptome analysis found that the expression of glpF,glpC,glpK,pfkA,argE,argF,argR,katG,sodA,sodB,and fumC in the two silent strains directly or indirectly related to bacterial intracellular survival changed significantly.The function of these genes is related to the acid resistance,osmotic pressure and oxidation resistance of bacteria.The results of biological phenotypic experiments showed that there was no difference between the growth of silent and wild strains under acidic stress and osmotic stress,but the antioxidant capacity of silent strains was lower than that of wild strains.The research data suggested that the two-component system EnvZ/OmpR mainly affects the ability of bacteria to survive in host phagocytes by regulating the antioxidant capacity of A.hydrophila.Based on the results,the following conclusions can be made:(1)The two-component system EnvZ/OmpR can regulate the pathogenicity of bacteria by regulating the type Ⅱ secretion system,chemotaxis,adhesion and biofilm formation of A.hydrophila.This indicates that the two-component system EnvZ/OmpR plays an important role in the pathogenesis of A.hydrophila B11.(2)After the two-component system EnvZ/OmpR was silenced,the expression of stress resistance and antioxidant genes related to intracellular survival of A.hydrophila changed significantly.Biological phenotype studies have shown that the two-component system EnvZ/OmpR mainly affects the ability of bacteria to survive in host phagocytes by regulating the antioxidant capacity of A.hydrophila.