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Abstract Pseudomonas plecoglossicida is a highly pathogenic bacterium for maricultured fish and causes serious losses. A transcriptional regulator gene RK21_RS10315 was found up-regulated during the whole infection process, which was confirmed by qRT-PCR. Five shRNA were designed to silence RK21_RS10315 gene, and the gene expression was reduced up to 96.1%. Compared with the counterpart infected with wild type strain, the infection of RK21_RS10315 -RNAi strain resulted in the death time delay, and 90% reduction in mortality of Epinephelus coioides , as well as the alleviation in the symptoms of E. coioides spleen. Moreover, compared with the fish infected with wild type strain, the infection of RK21_RS10315 -RNAi strain of P. plecoglossicida resulted in a significant change both in transcriptome of spleen of infected E. coioides and P. plecoglossicida. The KEGG analysis showed that genes of 16 immune pathways in E. coioides were affected by the silence of RK21_RS10315 of P. plecoglossicida. Among them, intestinal immune network for IgA production pathway and leukocyte transendothelial migration pathway were more prominent than other pathways. 19 euk-DEMs in these immune pathways had varying degrees of correlation with 19 pro-DEMs, and the expression of ipxA , grpE , yhbJ , truD and suhB from 19 pro-DEMs were predicted more related to RK21_RS10315 in P. plecoglossicida. Highlights • RNAi significantly reduced the content of RK21_RS10315 mRNA of P. plecoglossicida. • RNAi of RK21_RS10315 significantly reduced the pathogenicity of P. plecoglossicida to E. coioides. • Correlations in gene expression between euk-DEMs and pro-DEMs were affected in the pathogen-host interaction. [ABSTRACT FROM AUTHOR]

Abstract Pseudomonas plecoglossicida is an important and highly pathogenic bacterium for aquaculture and causes serious losses. The expression level of flgM was found to be significantly upregulated post-infection compared with in vitro results, which was confirmed by quantitative real-time PCR. RNAi significantly reduced the expression level of flgM mRNA of P. plecoglossicida. Compared with infection with the wild-type strain, infection with the flgM -RNAi strain resulted in a delay in death and a 75% reduction in the mortality of Epinephelus coioides , followed by alleviation of the symptoms in E. coioides spleen. Moreover, compared with infection with the wild-type strain, infection with the flgM -RNAi strain of P. plecoglossicida resulted in a significant change in the transcriptome of the spleens of infected E. coioides and P. plecoglossicida. KEGG analysis for E. coioides showed that genes of 17 immune pathways were most affected by flgM -RNAi of P. plecoglossicida. Among them, the expression of mhc2 , zap70 , rhoh , tlr2 , ca79a , hcst and cd32 in E. coioides spleen was predicted to be negatively related to flgM in P. plecoglossicida but positively related to genes involved in communication, metabolism and motility. Highlights • The pathogenicity of P. plecoglossicida to E. coioides was significantly reduced for the flgM -RNAi strain. •. RNAi of flgM significantly resulted in upregulation of immune pathways in E. coioides infected with P. plecoglossicida • The upregulated E. coioides immune genes co-expressed with P. plecoglossicida genes. [ABSTRACT FROM AUTHOR]

Background: Bacteria deliver effector proteins into the host cell via a secretory system that can directly act on the target to cause disease. As an important pipeline structural protein of the type VI secretion system (T6SS) complex, Hcp acts together with other virulence factors in the target cell. There is growing evidence that T6SS plays a key role in the pathogenic mechanism of APEC. However, the regulatory function played by the effector protein Hcp during its interaction with host cells is not clear. Here, tandem mass tag (TMT) analysis was used to quantify the proteins affected by increased expression of Hcp2a in DF-1 cells. Results: The host response was significantly different between the overexpression and null groups at the protein level. A total of 195 differentially expressed proteins (DEPs) were detected in the overexpression group (upregulated, n = 144, downregulated, n = 51). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological functions and pathways of differentially expressed proteins. The results showed that these DEPs were mainly enriched in RNA degradation, spliceosome, and mRNA surveillance pathways. Conclusions: This study suggests that Hcp2a, the effector protein of APEC, plays an important role in regulating mRNA splicing and protein quality control in DF-1 cells. These findings provide useful clues to elucidate the pathogenic mechanism of effector protein Hcp2a on host target cells. [ABSTRACT FROM AUTHOR]

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. • Pseudomonas plecoglossicida rpoD gene was silenced by RNA interference with an efficiency of 89.2%. • Silencing rpoD resulted in a 25% increase in the survival rate of E. coioides infected by P. plecoglossicida. • Silencing rpoD significant affected the immune response of infected E. coioides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Δ fliP) strain, fliP gene-complemented (C-Δ fliP) 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 Δ fliP 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 Δ fliP 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 Δ fliP strain mainly up-regulated the expression of inflammation related genes (IL-6 , IL-12 , IL-1β , 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. • fliP is a virulence gene of P. plecoglossicida. • Deletion of the fliP decreased bacterial load and attenuated the tissues damage in grouper. • Deletion of the fliP significantly affected immune response of grouper to P. plecoglossicida infection. [ABSTRACT FROM AUTHOR]