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Pseudomonas plecoglossicida is a gram-negative pathogenic bacterium that causes visceral white spot disease in several marine and aquaculture fish species, resulting in high mortality and severe financial loss. Based on previous RNA sequencing (RNA-seq), fliK gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that fliK may contribute to its bacterial pathogenicity. To investigate the role of fliK, four specific short hairpin RNAs (shRNAs) were designed and synthesized according to the fliK gene sequence, with three of the four mutants exhibiting a significant decrease in fliK gene expression in P. plecoglossicida. The shRNA-406 mutant with the maximum silencing efficiency (97.5%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain, silencing fliK in the fliK-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of fliK also resulted in a 95% increase in the survival rate, a 2-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface in infected orange-spotted groupers (Epinephelus coioides). In addition, fliK gene expression and pathogen load were significantly lower in the spleens of E. coioides infected with the fliK-RNAi strain than in those infected with the WT strain of P. plecoglossicida. RNA-seq of the spleens further revealed that fliK silencing significantly regulated the immune response of E. coioides during the pathogenic process. Compared with the WT-infected group, the differentially expressed genes (DEGs) in the fliK-RNAi-infected group were enriched in 344 and 345 KEGG pathways at 3 and 5 days post infection (dpi), respectively. Among these pathways, 21 immune system-related pathways were enriched, including the natural killer (NK) cell-mediated cytotoxicity, platelet activation, and Th17 cell differentiation signaling pathways. The NK cell-mediated cytotoxicity pathway was the most significantly enriched, which may enhance the host’s ability to remove pathogens and reduce inflammation. This study revealed the effects of the fliK gene in P. plecoglossicida pathogenicity and identified the main pathways involved in the immune response of E. coioides.

Pseudomonas plecoglossicida is the causative agent of “visceral white spot disease” in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.

Vibrio alginolyticus is a common opportunistic pathogen of fish, shrimp, and shellfish, and many diseases it causes can result in severe economic losses in the aquaculture industry. Causing host disease was confirmed by several virulence factors of V. alginolyticus. To date, there have been no reports on the effect of the pstS gene on its virulence regulation of V. alginolyticus. The virulence mechanism of target genes regulating V. alginolyticus is worthy of further study. Previous studies found that Fructus schisandrae (30 mg/mL) inhibited the growth of V. alginolyticus ND-01 (OD600 = 0.5) for 4 h, while the expressions of pstS and pstB were significantly affected by F. schisandrae stress. So, we speculated that pstS and pstB might be the virulence genes of V. alginolyticus, which were stably silenced by RNAi to construct the silencing strains pstS-RNAi and pstB-RNAi, respectively. After the expression of pstS or pstB gene was inhibited, the adhesion capacity and biofilm formation of V. alginolyticus were significantly down-regulated. The chemotaxis and biofilm formation ability of pstS-RNAi was reduced by 33.33% and 68.13% compared with the wild-type strain, respectively. Sequence alignment and homology analysis showed that pstS was highly conserved, which suggested that pstS played a vital role in the secretion system of V. alginolyticus. The pstS-RNAi with the highest silencing efficiency was selected for transcriptome sequencing. The Differentially Expressed Genes (DEGs) and GO terms were mapped to the reference genome of V. alginolyticus, including 1055 up-regulated genes and 1134 down-regulated genes. The functions of the DEGs were analyzed by GO and categorized into different enriched functional groups, such as ribosome synthesis, organelles, biosynthesis, pathogenesis, and secretion. These DEGs were then mapped to the reference KEGG pathways of V. alginolyticus and enriched in commonalities in the metabolic, ribosomal, and bacterial secretion pathways. Therefore, pstS and pstB could regulate the bacterial virulence of V. alginolyticus by affecting its adhesion, biofilm formation ability, and motility. Understanding the relationship between the expressions of pstS and pstB with bacterial virulence could provide new perspectives to prevent bacterial diseases.

Previously, the dual RNA-seq was carried out in a Pseudomonas plecoglossicida- Epinephelus coioides infection model to investigate the dynamics of pathogen-host interplay in vivo. ZnuC, a member of ZnuCBA Zn importer, was found transcriptionally up-regulated during infection. Thus, this study aimed to assess its role during the trade-off for Zn between host and P. plecoglossicida. ICP-MS analysis and fluorescent staining showed that Zn was withheld from serum and accumulated in the spleen, with increased Zn uptake in the Golgi apparatus of macrophages after infection. Additionally, growth assay, macrophage infection and animal infection after gene knockout / silencing revealed that znuC was necessary for growth in Zn-limiting conditions, colonization, intracellular viability, immune escape and virulence of P. plecoglossicida. Further analysis with dual RNA-seq revealed associations of host’s Zn nutritional immunity genes with bacterial Zn assimilation genes. IL6 and ZIP4 played key roles in this network, and markedly affected znuB expression, intracellular viability and immune escape, as revealed by gene silencing. Moreover, EMSA and GFP reporter gene analysis showed that Fur sensed changes in Fe concentration to regulate znuCBA in P. plecoglossicida. Jointly, these findings suggest a trade-off for Zn between host and P. plecoglossicida, while ZnuC is important for P. plecoglossicida Zn acquisition.

Diseases caused by Vibrio harveyi lead to severe economic losses in the aquaculture industry. Adhesion is an important disease-causing factor observed in bacteria with chemotactic activity. In our study, we measured the adhesion of V. harveyi by subjecting the bacteria to stress using Cu2+, Pb2+, Hg2+, and Zn2+. The genes responsible for chemotaxis (cheA, cheB, cheR, cheV, and cheY), which are also crucial for adhesion, were identified and silenced via RNAi. We observed that a decrease in chemotactic gene expression reduced the ability of the organism to demonstrate adhesion, motility, chemotaxis, and biofilm formation. Upon comparing the cheA-RNAi bacteria to the wild-type strain, we observed that the transcriptome of V. harveyi was significantly altered. Additionally, the expression of key genes and the adhesion ability were affected by the pH (pH of 5, 6, 7, 8, and 9), salinity (NaCl at concentrations of 0.8, 1.5, 2.5, 3.5, or 4.5%), and temperature (4, 15, 28, 37, and 44°C) of the medium. Based on these results, the following conclusions were made: (1) The chemotactic genes cheA, cheB, cheR, cheV, and cheY may regulate the adhesion ability of V. harveyi by affecting bacterial motility, and participate in the regulation of adhesion at different temperatures, salinities, and pH values; (2) stable silencing of cheA could alter the transcriptional landscape of V. harveyi and regulate the expression of genes associated with its adhesion mechanisms.

Abstract Aeromonas hydrophila B11 strain was isolated from diseased Anguilla japonica, which had caused severe gill ulcers in farmed eel, causing huge economic losses. EnvZ‐OmpR is a model two‐component system in the bacteria and is widely used in the research of signal transduction and gene transcription regulation. In this study, the ompR of A. hydrophila B11 strain was first silenced by RNAi technology. The role of ompR in the pathogenicity of A. hydrophila B11 was investigated by analyzing both the bacterial comparative transcriptome and phenotype. The qRT‐PCR results showed that the expression of ompR in the ompR‐RNAi strain decreased by 97% compared with the wild‐type strain. The virulence test showed that after inhibition of the ompR expression, the LD50 of A. hydrophila B11 decreased by an order of magnitude, suggesting that ompR is involved in the regulation of bacterial virulence. Comparative transcriptome analysis showed that the expression of ompR can directly regulate the expression of several important virulence‐related genes, such as the bacterial type II secretion system; moreover, ompR expression also regulates the expression of multiple genes related to bacterial chemotaxis, motility, adhesion, and biofilm formation. Further studies on the phenotype of A. hydrophila B11 and ompR‐RNAi also confirmed that the downregulation of ompR expression can decrease bacterial chemotaxis, adhesion, and biofilm formation.

Abstract Vibrio alginolyticus is one of the most important pathogens in mariculture and leading to heavy losses. After treatment with Cu2+, Pb2+, and low pH, the expression of oxidative phosphorylation pathway genes, including coxA, coxB, coxC, ccoN, ccoO, and ccoQ, was found commonly downregulated by RNA‐seq as well as quantitative real‐time PCR. RNAi significantly reduced the expression of coxA, coxB, coxC, ccoN, ccoO, and ccoQ in V. alginolyticus. Compared with the wild‐type strain, the adhesion abilities of RNAi strains of V. alginolyticus were significantly impaired, as well as their cytochrome C oxidase activity. ccoQ appeared to be more important in the regulation of bacterial adhesion in these target genes, while ccoO was relatively weak in the regulation of the adhesion. Meanwhile, the changes of temperature, salinity, pH, and starvation affected coxA, coxB, coxC, ccoN, ccoO, and ccoQ expression remarkably. These findings indicated that: the oxidative phosphorylation pathway is a critical regulator of adhesion in V. alginolyticus; coxA, coxB, coxC, ccoN, ccoO, and ccoQ regulate the bacterial adhesion in response to environmental changes such as temperature, salinity, pH, and starvation.

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 an important pathogen for aquaculture and causes high mortality in various marine fishes. Expression of sigX was found significantly up-regulated at 18°C than at 28°C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of sigX mRNA of P. plecoglossicida, whether in in vitro or in the spleen at all sampling time points. Compared with the wild-type strain, the infection of sigX-RNAi strain resulted in the onset time delay, and 20% reduction in mortality of Epinephelus coioides, as well as alleviates in the symptoms of E. coioides spleen. Compared with wild-type strain, the gene silence of sigX in P. plecoglossicida resulted in a significant change in transcriptome of infected E. coioides. The result of gene ontology and KEGG analysis on E. coioides showed that genes of serine-type endopeptidase and chemokine signaling pathway, coagulation and complement system, and intestinal immune network for IgA production pathway were mostly affected by sigX of P. plecoglossicida. Meanwhile, the immune genes were associated with different number of miRNA and lncRNA, and some miRNAs were associated with more than one gene at the same time. The results indicated that sigX was a virulent gene of P. plecoglossicida. The up-regulation of the immune pathways made E. coioides more likely to kill sigX-RNAi strain than the wild-type strain of P. plecoglossicida, while the immune genes were regulated by miRNA and lncRNA by a complex mode.

Pseudomonas plecoglossicida is a facultative pathogen that is associated with diseases of multiple fish, mainly at 15–20°C. Although fish disease caused by P. plecoglossicida has led to significant economic losses, the mechanisms of the temperature-dependent virulence are unclear. Here, we identify potential pathogenicity mechanisms and demonstrate the direct regulation of several virulence factors by temperature with transcriptomic and proteomic analyses, quantitative real-time PCR (qRT-PCR), RNAi, pyoverdine (PVD) quantification, the chrome azurol S (CAS) assay, growth curve measurements, a biofilm assay, and artificial infection. The principal component analysis, the heat map generation and hierarchical clustering, together with the functional annotations of the differentially expressed genes (DEGs) demonstrated that, under different growth temperatures, the animation and focus of P. plecoglossicida are quite different, which may be the key to pathogenicity. Genes involved in PVD synthesis and in the type VI secretion system (T6SS) are specifically upregulated at the virulent temperature of 18°C. Silencing of the PVD-synthesis-related genes reduces the iron acquisition, growth, biofilm formation, distribution in host organs and virulence of the bacteria. Silencing of the T6SS genes also leads to the reduction of biofilm formation, distribution in host organs and virulence. These findings reveal that temperature regulates multiple virulence mechanisms in P. plecoglossicida, especially through iron acquisition and T6SS secretion. Meanwhile, integration of transcriptomic and proteomic data provide us with a new perspective into the pathogenesis of P. plecoglossicida, which would not have been easy to catch at either the protein or mRNA differential analyses alone, thus illustrating the power of multi-omics analyses in microbiology.

Adhesion is an important virulence trait of Vibrio alginolyticus. Bacterial adhesion is influenced by environmental conditions; however, the molecular mechanism underlying this effect remains unknown. The expression levels of flrA, flrB and flrC were significantly downregulated in adhesion-deficient V. alginolyticus strains cultured under Cu2+, Pb2+, Hg2+ and low-pH stresses. Silencing these genes led to deficiencies in adhesion, motility, flagellar assembly, biofilm formation and exopolysaccharide (EPS) production. The expression levels of fliA, flgH, fliS, fliD, cheR, cheV and V12G01_22158 (Gene ID) were significantly downregulated in all of the RNAi groups, whereas the expression levels of toxT, ctxB, acfA, hlyA and tlh were upregulated in flrA- and flrC-silenced groups. These genes play a key role in the virulence mechanisms of most pathogenic Vibrio species. Furthermore, the expression of flrA, flrB and flrC was significantly influenced by temperature, salinity, starvation and pH. These results indicate that (1) flrA, flrB and flrC are important for V. alginolyticus adhesion; (2) flrA, flrB and flrC significantly influence bacterial adhesion, motility, biofilm formation and EPS production by controlling expression of key genes involved in those phenotypes; and (3) flrA, flrB and flrC regulate adhesion in the natural environment with different temperatures, pH levels, salinities and starvation time.Emerging Microbes & Infections (2016) 5, e85; doi:10.1038/emi.2016.82; published online 3 August 2016

In the present study, Larimichthys crocea and Pseudomonas plecoglossicida were selected as a host-pathogen interaction model for teleosts and prokaryotic pathogens. Five shRNAs were designed and synthesized to silence the fliA gene, all of which resulted in pronounced reductions in fliA mRNA; the mutant strain with the best silencing efficiency of 92.16% was chosen for subsequent analysis. A significant decrease in motility, intracellular survival and escape was observed for the fliA-RNAi strain of P. plecoglossicida, whereby silencing of the fliA gene led to a 30% decrease in mortality and a four-day delay in the onset of infection in L. crocea. Moreover, silencing of P. plecoglossicida fliA resulted in a significant change in both the pathogen and host transcriptome in the spleens of infected L. crocea. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of pathogen transcriptome data showed that silencing fliA resulted in downregulation of 18 flagellum-related genes; KEGG analysis of host transcriptome data revealed that infection with the fliA-RNAi strain caused upregulation of 47 and downregulation of 106 immune-related genes. These pathogen-host interactions might facilitate clearance of P. plecoglossicida by L. crocea, with a significant decrease in fliA-RNAi P. plecoglossicida strain virulence in L. crocea.