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Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one‐sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV‐RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV‐RNAi strain failed to develop visible nodules at 5–8 dpi, with the swelling gradually disappearing. The burdens of clpV‐RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV‐RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu2+, Pb2+, Hg2+ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control. [ABSTRACT FROM AUTHOR]

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]

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H2O2. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages. [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is associated with multiple fish diseases, and temperature is one of the most important environmental factors related to its outbreak. To elucidate the influence of temperature variation on the pathogen, the global metabolomics of P. plecoglossicida (NZBD9) were analysed at the virulent (18°C) and avirulent (12°C and 28°C) temperatures. The result showed that the levels of Phosphoric acid, Tyrosine, Spermidine and Sucrose were significantly reduced，while Itaconic acid, Glucaric acid and Isomaltose were increased in P. plecoglossicida at 18°C. These metabolic adjustments assist P. plecoglossicida to survive in adverse environments, proliferate in the host, colonize and resist host immune clearance during the initial steps of infection. The results suggested that L321_03626 and L321_18122 genes played a key role in the regulation of these metabolic adaptions and thus regulated P. plecoglossicida virulence at virulent temperature, which was proved by further gene silencing and artificial infection. The present study, for the first time, determines the P. plecoglossicida metabolomic responses to temperature variation, which is helpful to explore its pathogenic mechanism and provides reference for disease control. [ABSTRACT FROM AUTHOR]