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Aeromonas salmonicida is a common pathogenic bacterial species found in both freshwater and marine fish, leading to significant economic losses in the aquaculture industry. YidC is an accessory to SecYEG and is essential for the SecYEG transporter to insert into the bacterial membrane. However, the roles of the yidC gene on the host immune response remain unclear. Here, we compared the pathogenicity of yidC gene-deleted (ΔyidC) strain and wild-type (SRW-OG1) strain of mesophilic A. salmonicida to Orange-spotted grouper (Epinephelus coioides), and explored the impacts of yidC gene on the immune response of E. coioides to mesophilic A. salmonicida infection by using Red/ET recombineering. In this study, the E. coioides in the Secondary infected group had a 53.9 % higher survival rate than those in the Primary infected group. In addition, the adhesion ability of ΔyidC strain decreased by about 83.36 % compared with that of the wild-type (SRW-OG1) strain. Further comparison of the biological phenotype of SRW-OG1 and ΔyidC revealed that this yidC gene could regulate the expression of genes related to iron metabolism and have no effect on bacterial growth under the limited iron concentration. In the low concentration of Fe 3+ and Fe 2+ environment, SRW-OG1 can obtain iron ions by regulating yidC. Based on the above results, yidC gene contributed to the pathogenicity of mesophilic A. salmonicida to E. coioides, deletion of yidC gene promoted the inflammation and immune response of E. coioides to mesophilic A. salmonicida infection., (Copyright © 2024. Published by Elsevier Ltd.)

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 Zn 2+ and Mg 2+ 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 H 2 O 2 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 Mg 2+ homeostasis in fish macrophages., (© 2022 John Wiley & Sons Ltd.)

Large yellow croaker (Larimichthys crocea) is an economical important farmed fish in China. "Visceral White Spot Disease" caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L. crocea in recent years and resulted in heavy economy lossess. The dual RNA-seq results of previous study showed that the expression of clpV gene in P. plecoglossicida was significantly up-regulated during infection. RNAi significantly reduced the expression of clpV in P. plecoglossicida with maximum silencing efficiency of 96.1%. Compared with the wild type strain, infection of clpV-RNAi strain resulted in a delayed onset time and a 25% reduction in mortality of L. crocea, as well as lessening the symptoms of the spleen. The results of dual RNA-seq of L. crocea infected by clpV-RNAi strain of P. plecoglossicida changed considerably, compared with the counterpart infected with the wild strain. The KEGG enrichment analysis showed that Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway and MAPK signaling pathway of L. crocea were most affected by the silence of clpV in P. plecoglossicida. RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the "Sec secretion system" were significantly downregulated. This downregulation of "Sec secretion system" genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. The expression of an ABC transporter gene-L321_23611 of P. plecoglossicida at 18 °C was found significant higher than those at 28 °C by RNA-seq and qRT-PCR. RNAi significantly reduced the content of L321_23611 mRNA in P. plecoglossicida with a maximal decrease of 89.2%. Compared with the wild type strain, the infection of L321_23611-RNAi strain resulted in the reduction in mortality and the onset time delay of a kind of marine teleosts, Epinephelus coioides. The results of dual RNA-seq showed that the RNAi of L321_23611 resulted in a significant change in both pathogen and host transcriptome in the spleens of infected E. coioides. The result of GO and KEGG analysis from dual RNA-seq data showed both host genes of chemokine signaling pathway, coagulation and complement system, hematopoietic cell lineage pathway as well as hemoglobin complex GO term and pathogenic genes of bacterial-type flagellum-dependent cell mortality GO term and flagellar assembly, biosynthesis of amino acids and lysine biosynthesis systems pathways were mainly affected by L321_23611 gene of P. plecoglossicida. The results indicated that: 1. ABC transporter gene-L321_23611 was a virulent gene of P. plecoglossicida. 2. Both the activation of the host immune pathways and depression of pathogenic virulence-related pathways facilitated E. coioides to remove L321_23611-RNAi strain than the wild type strain of P. plecoglossicida., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 John Wiley & Sons Ltd.)

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of "white nodules" disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG -RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides , and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides , dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B , and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG -dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG -RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida , while CELA2, TRY, CPA1, CPA2 , and CPB1 were important targets for P. plecoglossicida attacking to the host.

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., (© 2019 John Wiley & Sons Ltd.)

The incidence of Vibrio alginolyticus infections has increased in recent years due to the influence of climate change and rising sea temperature. Vibrio virulence regulatory RNA 1 (Vvrr1) is a newly found noncoding RNA (ncRNA) predicted to be closely related to the adhesion ability of V. alginolyticus based on the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1-overexpressing strains and using the proteome sequencing technology. Pyruvate kinase I ( pykF ) gene was predicted to be a chief target gene of Vvrr1 involved in virulence regulation. The adhesion ability, biofilm formation and virulence were significantly reduced in the Vvrr1-overexpressing and the pykF -silenced strain compared with the wild strains. Similar to the overexpression of Vvrr1, the silencing of pykF also reduced the expression level of virulence genes, such as ndk , eno , sdhB , glpF , and cysH . Meanwhile, by constructing the " pykF-GFP " fusion expression plasmid and using the GFP reporter gene analysis in Escherichia coli , the fluorescence intensity of the strain containing Vvrr1 whole ncRNA sequence vector was found to be significantly weakened. These indicated that Vvrr1 participated in the virulence regulation mechanism of V. alginolyticus by interacting with the virulence gene pykF .

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adhesion capability to fish mucus, which can be affected by environmental conditions, is considered to be a key virulence factor of Vibrio alginolyticus although the molecular mechanism is still unclear. In the present study, V. alginolyticus was treated with stress conditions including Cu(2+) (50 mg/L), Pb(2+) (100 mg/L), Hg(2+) (50 mg/L) and low pH (pH 5). We found these stress treatments were capable of reducing the adhesion of V. alginolyticus, while the expression levels of multiple genes were significantly changed according to the results of high throughput sequencing. The expression of randomly selected genes was confirmed by QPCR, which reinforced the reliability of the sequencing data. Ontology assignments and KEGG pathway analysis indicated that stress treatments affect pathways that may be related to adhesion. Our results identified genes which might play a key role in the adhesion process of V. alginolyticus, which could lay a foundation for further functional analysis of these genes in the process of adhesion. As these genes were sensitive to environmental factors, this may explain why the adhesion process can be influenced by environmental factors.

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]