Comparative transcriptome analysis explored the molecular mechanisms of a luxR-type regulator regulating intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila is not a traditional intracellular bacterium. However, previous studies revealed that pathogenic A. hydrophila B11 could temporarily survive for at least 24 h in fish phagocytes, and the regulation of intracellular survival in bacteria was associated with regulators of the LuxR-type. The mechanisms of luxR08110 on the A. hydrophila's survival in macrophages were investigated using comprehensive transcriptome analysis and biological phenotype analysis in this study. The results showed that after luxR08110 was silenced, the intracellular survival ability of bacteria was significantly diminished. Comparative transcriptome analysis revealed that luxR08110 was a critical regulator of A. hydrophila, which regulated the expression of over 1200 genes, involving in bacterial flagellar assembly and chemotaxis, ribosome, sulphur metabolism, glycerolipid metabolism, and other mechanisms. Further studies confirmed that after the inhibition of expression of luxR08110, the motility, chemotaxis and adhesion of A. hydrophila significantly decreased. Moreover, compared with the wild-type strain, the survival rates of silencing strain were all considerably reduced under both H2O2 and low pH stress conditions. According to both transcriptome analysis and phenotypic tests, the luxR08110 of A. hydrophila could act as global regulator in bacteria intracellular survival. This regulator regulated intracellular survival of A. hydrophila mainly through two ways. One way is to regulate bacterial flagellar synthesis and further affects the motility, chemotaxis and adhesion of bacteria. The other way is to regulate sulphur and glycerolipid metabolisms, thus affecting bacterial energy production and the ability to resist environmental stress.

flgC gene is involved in the virulence regulation of Pseudomonas plecoglossicida and affects the immune response of Epinephelus coioides.

As a pathogen of cultured teleosts, Pseudomonas plecoglossicida has caused significant economic losses. flgC plays an important role in encoding flagellar basal-body rod proteins. Our previous studies revealed the high expression of P. plecoglossicida flgC in infected Epinephelus coioides. To explore the role of flgC in the virulence of P. plecoglossicida and the immune response of E. coioides to the infection of P. plecoglossicida, flgC gene of P. plecoglossicida was knocked down by RNA interference (RNAi). The results showed that the flgC gene in all four mutants of P. plecoglossicida was significantly knocked down, and the mutant with the best knockdown efficiency of 94.3% was selected for subsequent studies. Compared with the NZBD9 strain of P. plecoglossicida, the flgC-RNAi strain showed a significantly decrease in chemotaxis, motility, adhesion, and biofilm formation. Furthermore, compared with the E. coioides infected with the NZBD9 strain, the infection of flgC-RNAi strain resulted in the infected E. coioides a 1.5-day delay in the time of first death and an 80% increase in survival rate, far fewer white nodules upon the spleen surfaces, and lower pathogen load in the spleens. RNAi of flgC significantly influenced the metabolome and transcriptome of the spleen in infected E. coioides. KEGG enrichment analysis exhibited that the Toll-like receptor signaling pathway was the most enriched immune pathway; the most significantly enriched metabolic pathways were associated with Linoleic acid metabolism, Choline metabolism in cancer, and Glycerophospholipid metabolism. Further combined analysis of transcriptome and metabolome indicated significant correlations among pantothenate and CoA biosynthesis, beta-alanine metabolism, lysosome metabolites, and related genes. These results suggested that flgC was a pathogenic gene of P. plecoglossicida; flgC was associated with the regulation of chemotaxis, motility, biofilm formation, and adhesion; flgC influenced the immune response of E. coioides to the infection of P. plecoglossicida.

Pseudomonas plecoglossicida fliP gene affects the immune response of Epinephelus fuscoguttatus ♀×Epinephelus lanceolatus ♂ to infection.

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease in a variety of teleosts. The protein encoded by fliP gene is involved in the assembly of bacterial flagella, which plays a vital role in bacterial pathogenicity. However, the roles of the fliP gene on the host immune response remain unclear. Here, we compared the pathogenicity of fliP gene-deleted (ΔfliP) strain, fliP gene-complemented (C-ΔfliP) strain and wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), and explored the impacts of fliP gene on the immune response of hybrid grouper to P. plecoglossicida infection by using RNA-seq. In this study, the grouper in the ΔfliP strain-infected group had a 30% higher survival rate than those in the NZBD9 strain-infected group. In addition, the deletion of fliP gene decreased bacterial load in the spleen, intestine, liver as well as head kidney of hybrid grouper and the tissues damage were weakened. Moreover, the infection of hybrid grouper spleen by the ΔfliP strain induced 1,189 differential expression genes compared with the counterpart infected by NZBD9 strain. KEGG enrichment analysis showed that 9 immune-related pathways, 5 signal transduction pathways, and 3 signaling molecules and interaction pathways were significantly enriched. qRT-PCR analysis revealed that the ΔfliP strain mainly up-regulated the expression of inflammation related genes (IL-6, IL-12, IL-1ß, IL-10, CXCL8, CXCL10) and immune regulation related genes (TLR2, P65, MyD88, P85, AKT), but down-regulated the expression of cell death related genes (FoxO1, Bim, PLK2 and LDHA) during infection. Based on the above results, fliP gene contributed to the pathogenicity of P. plecoglossicida to hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂), deletion of fliP gene promoted the inflammation and immune response of hybrid grouper to P. plecoglossicida infection, which accelerating host clearance of pathogen and reducing tissue damages.

The involvement of the T6SS vgrG gene in the pathogenicity of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida, the causative agent of white spot disease of large yellow croaker, has caused serious economic losses to the aquaculture industry. The type VI secretion system (T6SS) is a significant virulence system widely distributed among Gram-negative bacteria. VgrG, a structural and core component of T6SS, is crucial to the function of T6SS. To explore the biological profiles mediated by vgrG gene and its effects on the pathogenicity of P. plecoglossicida, the vgrG gene deletion (ΔvgrG) strain and complementary (C-ΔvgrG) strain were constructed and the differences in pathogenicity and virulence-related characteristics between different strains were analysed. The results showed that vgrG gene deletion significantly affected the virulence-related characteristics of P. plecoglossicida, including chemotaxis, adhesion, and biofilm formation. In addition, the LD50 of ΔvgrG strain was nearly 50-fold higher than that of the NZBD9 strain. Transcriptome data analysis suggested that the vgrG gene may affect the virulence of P. plecoglossicida by regulating the quorum sensing pathway to inhibit the secretion of virulence factors and affect biofilm formation. Besides, deletion of the vgrG gene may reduce bacterial pathogenicity by affecting bacterial signal transduction processes and the ability to adapt to chemotactic substances.

The role and mechanisms of rstA in the intracellular survival of fish pathogenic Aeromonas hydrophila.

In this study, RNAi technology was used to silence the gene rstA in Aeromonas hydrophila. The strain rstA-RNAi displayed significant decrease in intracellular survival compared with that of the wild-type strain B11. Transcriptome analysis explored that the expression of some important anti-stress protein genes was significantly upregulated in rstA-RNAi compared with the wild-type strain, while the expression of the genes related to iron acquisition and type VI secretion system was significantly downregulated. Further study found that under low pH and H2 O2 stress, the anti-stress protein genes were expressed at a low level in rstA-RNAi, the growth ability of rstA-RNAi was also significantly lower than that of wild-type strain. The results also displayed that with the fluctuation of iron concentration, the expression of some genes related to iron acquisition remained at a low level in rstA-RNAi, and the growth ability of rstA-RNAi was lower than that of the wild-type strain under the same culture conditions, indicating rstA can regulate iron acquisition and further affect the bacteria growth. The adhesion ability of rstA-RNAi to fish macrophages was reduced, suggesting rstA may be also affect the formation of type VI secretion system of A. hydrophila.

Function of the rpoD gene in Pseudomonas plecoglossicida pathogenicity and Epinephelus coioides immune response.

Pseudomonas plecoglossicida is a Gram-negative pathogenic bacterium that causes visceral white spot disease in several marine fish species, resulting in high mortality and financial loss. Based on previous RNA sequencing (RNA-seq) results, rpoD gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that rpoD may contribute to bacterial pathogenicity. To investigate the role of this gene, five specific short hairpin RNAs (shRNAs) were designed and synthesized based on the rpoD gene sequence, with all five mutants exhibiting a significant decrease in rpoD gene expression in P. plecoglossicida. The mutant with the highest silencing efficiency (89.2%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain NZBD9, silencing rpoD in the rpoD-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of rpoD also resulted in a 25% increase in the survival rate, a one-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface of infected orange-spotted groupers (Epinephelus coioides). In addition, rpoD expression and pathogen load were significantly lower in the spleens of E. coioides infected with the rpoD-RNAi strain than with the WT strain of P. plecoglossicida. We performed RNA-seq of E. coioides spleens infected with different P. plecoglossicida strains. Results showed that rpoD silencing in P. plecoglossicida led to a significant change in the infected spleen transcriptomes. In addition, comparative transcriptome analysis showed that silencing rpoD caused significant changes in complement and coagulation cascades and the IL-17 signaling pathway. Thus, this study revealed the effects of the rpoD gene on P. plecoglossicida pathogenicity and identified the main pathway involved in the immune response of E. coioides.

The contribution of exbB gene to pathogenicity of Pseudomonas plecoglossicida and its interactions with Epinephelus coioides.

To study the roles of the exbB gene in Pseudomonas plecoglossicida during interactions with Epinephelus coioides, five short hairpin RNAs (shRNAs) were designed and synthesized to silence the exbB gene in P. plecoglossicida which resulted in significant reductions in exbB mRNA expression. The mutant with the best silencing efficiency (89.3%) was selected for further study. Silencing exbB in the exbB-RNA interference (RNAi) strain resulted in a 70% increase in the survival rate and a 3-day delay in the onset of infection in E. coioides. Silencing of the exbB gene also resulted in a significant decrease in the number of white spots on the spleen surface and in the spleen pathogen load. The results of dual RNA-seq showed that exbB silencing in P. plecoglossicida also resulted in a significant change in both the pathogen and host transcriptomes in the spleens of infected E. coioides. Comparative transcriptome analysis showed that silencing exbB caused significant changes in multiple signaling molecules and interaction- and immune system-related genes in E. coioides. Gene silencing also resulted in the differential expression of flagellar assembly and the bacterial secretion system in P. plecoglossicida during the infection period, and most of the DEGs were down-regulation. These host-pathogen interactions may make it easier for E. coioides to eliminate the exbB-RNAi strain of P. plecoglossicida, suggesting a significant decrease in the pathogenicity of this strain. These results indicated that exbB was a virulence gene of P. plecoglossicida which contributed a lot in the pathogen-host interactions with E. coioides.

The role and mechanisms of the two-component system EnvZ/OmpR on the intracellular survival of Aeromonas hydrophila.

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 H2 O2 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.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides.

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

Integration of RNA-seq and RNAi reveals the contribution of znuA gene to the pathogenicity of Pseudomonas plecoglossicida and to the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 105  cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.

Time-resolved dual RNA-seq of tissue uncovers Pseudomonas plecoglossicida key virulence genes in host-pathogen interaction with Epinephelus coioides.

Bacterial pathogen-host interactions are highly dynamic, regulated processes that have been primarily investigated using in vitro assays. The dynamics of bacterial pathogen-host interplay in vivo are poorly understood. Using time-resolved dual RNA-seq in a Pseudomonas plecoglossicida-Epinephelus coioides infection model, we observed that bacterial genes encoding classical virulence factors and host genes involved in immune regulation were dynamically expressed during infection. Using network inferencing, we were able to predict interspecies regulatory networks linking bacterial virulence genes to host immune genes. Together with gene co-expression network analysis of the pathogen, secY was predicted to be a key virulence gene for P. plecoglossicida pathogenicity in the host, fliN was predicted to be a less important virulence gene. The results of bioinformatics prediction were confirmed by animal infection experiments. Our work provides the first paradigm to study dynamic alterations of bacterial pathogen and host interactions based on the elucidation of time-resolved interactive transcriptomes in vivo, and may be developed into a novel and universal method for revealing the true complexity of the bacterial infection process.

Integration of RNA-seq and RNAi provides a novel insight into the immune responses of Epinephelus coioides to the impB gene of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a Gram-negative bacterium that causes visceral white spot disease in Epinephelus coioides and leads to severe aquatic economic losses. The RNA-seq results of a previous study showed that the expression of the impB gene in P. plecoglossicida was significantly upregulated during infection. Four shRNAs were designed and synthesized to silence the impB gene in P. plecoglossicida, and the maximum silencing efficiency was 95.2%. Intraperitoneal injection of the impB-RNAi strain of P. plecoglossicida did not cause E. coioides death, and the spleens of infected fish did not show significant clinical symptoms. Although the injection of the mutant strain increased the antibody titer in E. coioides serum, it could not effectively protect E. coioides against wild strain infection. Compared with E. coioides infected with the wild type strain, the RNA-seq results for E. coioides infected with the impB-RNAi strain differed greatly. The KEGG enrichment analysis showed that key genes of the chemokine signalling pathway of E. coioides were downregulated by the silencing of impB in P. plecoglossicida. Infection with the impB-RNAi strain of P. plecoglossicida through injection did not produce good immune protection against E. coioides. The present study provides a novel insight into the immune responses of E. coioides to the impB gene of P. plecoglossicida.

Role of LuxR-type regulators in fish pathogenic Aeromonas hydrophila.

LuxR-type transcriptional factors are essential in many bacterial physiological processes. However, there have been no reports on their roles in Aeromonas hydrophila. In this study, six stable silent strains were constructed using shRNA. Significant decreases in the expression levels of luxR05 , luxR08 , luxR19 , luxR11 , luxR164 and luxR165 were shown in their respective strains by qRT-PCR. The luxR05 -RNAi and luxR164 -RNAi exhibit the most significant changes in sensitivity to kanamycin and gentamicin. The luxR05 -RNAi showed minimum biofilm formation and the least motility, while luxR164 -RNAi showed minimum biofilm formation, adhesion, growth and extracellular protease activity compared to the wild-type strain. In summary, the results of this paper suggest that all six luxR genes are involved in multiple physiological processes in A. hydrophila and that the roles of luxR05 and luxR164 are highly significant. The sensitivity of luxR05 -RNAi and luxR164 -RNAi to drugs may be closely related to biofilm formation. The luxR05 may play an important role in the pathogenicity of A. hydrophila by regulating the movement, adhesion and biofilm formation of bacteria, whereas luxR164 may be involved in similar functions by regulating bacterial adhesion, extracellular enzyme activity and growth. These results help further our understanding of the drug resistance and pathogenesis of A. hydrophila.

Dual RNA-Seq uncovers the function of an ABC transporter gene in the host-pathogen interaction between Epinephelus coioides and Pseudomonas plecoglossicida.

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.

Integrated dual RNA-seq and dual iTRAQ of infected tissue reveals the functions of a diguanylate cyclase gene of Pseudomonas plecoglossicida in host-pathogen interactions with Epinephelus coioides.

The interactions between host and pathogen is exceedingly complex, which involves alterations at multiple molecular layers. However, research to simultaneously monitor the alterations of transcriptome and proteome between a bacterial pathogen and aquatic animal host through integrated dual RNA-seq and dual iTRAQ of tissue during infection is currently lacking. The important role of a diguanylate cyclase gene (L321_RS15240) in pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides was suggested by previous dual RNA-seq of our lab. Then L321_RS15240-RNAi strains of P. plecoglossicida were constructed with pCM130/tac, and the mutant with the best silencing effect was selected for follow-up study. The RNAi of L321_RS15240 resulted in a significant decrease in bacterial virulence of P. plecoglossicida. The E. coioides spleens infected by wild type strain or L321_RS15240-RNAi strain of P. plecoglossicida were subjected to dual RNA-seq and dual iTRAQ, respectively. The results showed that: RNAi of L321_RS15240 led to 1)alterations of host transcriptome associated with complement and coagulation cascades, ribosome, arginine and proline metabolism, and oxidative phosphorylation; 2)high expression of host proteins which related to phagosome and metabolism responses (metabolism of glutathione, amino sugar and nucleotide sugar); 3)the highly differentially expression of host lncRNAs and miRNAs. The differentially expressed proteins and mRNAs of pathogen were different after infection, but the functions of these proteins and mRNAs were mainly related to metabolism and virulence. This study provides a new insight to comprehensively understand the gene functions of pathogens and hosts at multiple molecular layers during in vivo infection.

Dual RNA-seq uncovers the immune response of Larimichthys crocea to the secY gene of Pseudomonas plecoglossicida from the perspective of host-pathogen interactions.

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.

Mechanistic insight into the roles of Pseudomonas plecoglossicida clpV gene in host-pathogen interactions with Larimichthys crocea by dual RNA-seq.

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.

The role of sodA and sodB in Aeromonas hydrophila resisting oxidative damage to survive in fish macrophages and escape for further infection.

Several bacteria have been defined as extracellular pathogens; however, in recent years, it has been confirmed that they have the ability to survive and escape the attack of host phagocytes, thus causing further infection. Previous studies have shown that Aeromonas hydrophila could survive in fish macrophages; however, the mechanism remains unknown. In this study, sodA and sodB of the strain A. hydrophila B11 were stable silenced by shRNA. The survival rates of intracellular sodA-RNAi and sodB-RNAi decreased by 91.8% and 74.9% and the immune escape rates decreased by about 32% and 92% respectively. At the same time, reactive oxygen species (ROS) in fish macrophages that phagocytosed sodA-RNAi and sodB-RNAi increased by 40% and 32.6%, respectively, compared to those of macrophages that phagocytosed the wild-type strain. Compared to sodA, the expression of sodB predominates in A. hydrophila without oxidative stress; however, when exposed to oxidative stress, the magnitude of up-regulation of sodA expression is significantly higher than that of sodB. With increased of methyl viologen concentration, the survival rates of sodA-RNAi and sodB-RNAi were significantly decreased. The expressions of sodA and sodB did not affect the growth of A. hydrophila without oxidative stress, but the inhibition of sodA and sodB expression led to a slight decrease in bacterial growth under oxidative stress. These results indicated that (1) sodA and sodB play an important role in the process of bacterial resistance to ROS damage in host phagocytic cells, allowing them to survive or even escape fish macrophages; (2) the sodB expression was dominant in A. hydrophila without oxidative stress, the sodA expression was up-regulated more significantly under oxidative stress, and sodA and sodB contributed equally to the process of bacterial resistance to ROS; (3) sodA and sodB complement each other and cooperate in the process of intracellular survival of bacteria to protect against ROS damage.

The effect of a LysR-type transcriptional regulator gene of Pseudomonas plecoglossicida on the immune responses of Epinephelus coioides.

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. It was determined with RNA-seq that the expression of a LysR-type transcriptional regulator gene (L321_20267) of P. plecoglossicida at 18 °C was significantly higher than that at 28 °C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of L321_20267 mRNA in P. plecoglossicida, with a maximal decrease of 90.63%. Compared with the wild-type strain, infection with the L321_20267-RNAi strain resulted in a 50% reduction in mortality and an onset time delay of Epinephelus coioides, as well as alleviation of the symptoms in E. coioides spleens. Compared with the wild-type strain of P. plecoglossicida, the L321_20267-RNAi strain resulted in a significant change in the spleen transcriptome of infected E. coioides. The results of GO and KEGG analysis showed that genes of serine hydrolase activity, the antigen processing and presentation pathway, the B cell receptor signalling pathway and the chemokine signalling pathway were most affected by the L321_20267 gene of P. plecoglossicida. Meanwhile, the immune genes were related to different numbers of miRNAs and lncRNAs, and some miRNAs were related to more than one gene. The results indicated that 1. L321_20267 is a virulence gene of P. plecoglossicida; 2. the upregulation of the immune pathways facilitated E. coioides to remove the L321_20267-RNAi strain compared with the wild-type strain of P. plecoglossicida; and 3. the immune genes were regulated by miRNA and lncRNA in a complex manner.

Silencing of cyt-c4 led to decrease of biofilm formation in Aeromonas hydrophila.

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11 has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4 gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila. ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.