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Aeromonas hydrophila infections are common in aquaculture. Our previous studies found that the A. hydrophila B11 strain can survive in fish macrophages for at least 24 h and the two‐component system EnvZ/OmpR may be involved in intracellular survival. To reveal the role and mechanism of the two‐component system EnvZ/OmpR in intracellular survival of A. hydrophila, the genes of envZ/ompR were silenced by shRNAi. The results showed that the survival rates of the envZ‐RNAi and ompR‐RNAi strains were only 2.05% and 3.75%, respectively, which were decreased by 91% and 83.6% compared with that of the wild‐type strain. The escape ability of envZ‐RNAi and ompR‐RNAi was also decreased by 51.4% and 19.7%, respectively. The comparative transcriptome analysis revealed that the functional genes directly related to bacterial intracellular survival mainly included the genes related to anti‐stress capacity, and the genes related to Zn2+ and Mg2+ transport. Further research confirmed that two‐component system EnvZ/OmpR can regulate the expression of the important molecular chaperones, such as groEL, htpG, dnaK, clpB and grpE. The expression of these molecular chaperones in wild‐type strain was up‐regulated with the increase in H2O2 concentrations, while the expression of these molecular chaperones in silent strains did not change significantly. Cells that phagocytosed wild‐type strain had higher ROS content than cells that phagocytosed silent strains. Two‐component system EnvZ/OmpR could also regulate zinc transporter (znuA, znuB, znuC) and zinc efflux protein (zntA) to maintain zinc homeostasis in cells, thus affecting the ability of bacteria to survive in phagocytes. Moreover, two‐component system EnvZ/OmpR could affect the growth and intracellular survival of A. hydrophila by regulating the expression of MgtA, MgtC and MgtE and participating in bacterial Mg2+ homeostasis in fish macrophages. [ABSTRACT FROM AUTHOR]

The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A . hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A . hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A . hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas . KatG of A . hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG -RNAi was significantly reduced. The survival rate of intracellular KatG -RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG -RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A . hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A . hydrophila is conserved; (2) KatG helped A . hydrophila to survive in fish macrophages by eliminating the harm of intracellular H 2 O 2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A . hydrophila could escape from host macrophages for further infection, in this process KatG also played important role. [ABSTRACT FROM AUTHOR]