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• A significant decrease in gene expression of adhesion-deficient V. harveyi. • fliA , fliR , and flrB were responsible for flagellation and silenced via RNAi. • fliA , fliR , and flrB have important roles in the adhesion of V. harveyi. • fliA , fliR , flrB regulate bacterial adhesion by affecting its motility, biofilm formation. • fliA , fliR , flrB regulate adhesion ability of V. harveyi in different environments. Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA , fliR , and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS , fliD , flgH , and flrC were significant down-regulated in fliR -RNAi, flrB -RNAi, and fliA -RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA , fliR , and flrB. Also, the expression of fliA , fliR , and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA , fliR , and flrB have important roles in the adhesion of V. harveyi ; (2) fliA , fliR , and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA , fliR , and flrB can regulate adhesion ability of V. harveyi in different environments. [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]