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Vibrio harveyi is a normal flora in natural marine habitats and a significant opportunistic pathogen in marine animals. This bacterium can cause a series of lesions after infecting marine animals, in which muscle necrosis and ulcers are the most common symptoms. This study explored the adaptation mechanisms of V. harveyi from the seawater environment to host fish muscle environment. The comprehensive transcriptome analysis revealed dramatic changes in the transcriptome of V. harveyi during its adaptation to the host fish muscle environment. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, flagellar assembly, oxidative phosphorylation, bacterial chemotaxis, and two-component systems play crucial roles in V. harveyi's adaptation to host fish muscle. A comparison of biological phenotypes revealed that V. harveyi displayed a significant increase in flagellar length, swimming, twitching, chemotaxis, adhesion, and biofilm formation after induction by host fish muscle, and its dominant amino acids, especially bacterial chemotaxis induced by host muscle, Ala and Arg. It could be speculated that the enhancement of bacterial chemotaxis induced by amino acids plays a key role in the adaptation of V. harveyi from seawater to the muscle of the host fish.
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Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.
Assuntos
Doenças dos Peixes , Flagelos , Regulação Bacteriana da Expressão Gênica , Pseudomonas , Pequeno RNA não Traduzido , Pseudomonas/patogenicidade , Pseudomonas/genética , Pseudomonas/fisiologia , Virulência/genética , Animais , Doenças dos Peixes/microbiologia , Pequeno RNA não Traduzido/genética , Flagelos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Bacteriano/genética , Sistemas de Secreção Tipo III/genética , Bass , Infecções por Pseudomonas/imunologiaRESUMO
Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1ß, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.
Assuntos
Doenças dos Peixes , Infecções por Pseudomonas , Pseudomonas , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/genética , Pseudomonas/patogenicidade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Infecções por Pseudomonas/microbiologia , Bass/imunologia , Bass/microbiologia , Bass/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genética , Transcriptoma , Perfilação da Expressão Gênica , Proteínas de Peixes/genética , Proteínas de Peixes/imunologiaRESUMO
WRKY transcription factors constitute one of the largest plant-specific gene families, regulating various aspects of plant growth, development, physiological processes, and responses to abiotic stresses. This study aimed to comprehensively analyze the WRKY gene family of yam (Dioscorea opposita Thunb.), to understand their expression patterns during the growth and development process and their response to different treatments of yam and analyze the function of DoWRKY71 in detail. A total of 25 DoWRKY genes were identified from the transcriptome of yam, which were divided into six clades (I, IIa, IIc, IId, IIe, III) based on phylogenetic analysis. The analysis of conserved motifs revealed 10 motifs, varying in length from 16 to 50 amino acids. Based on real-time quantitative PCR (qRT-PCR) analysis, DoWRKY genes were expressed at different stages of growth and development and responded differentially to various abiotic stresses. The expression level of DoWRKY71 genes was up-regulated in the early stage and then down-regulated in tuber enlargement. This gene showed responsiveness to cold and abiotic stresses, such as abscisic acid (ABA) and methyl jasmonate (MeJA). Therefore, further study was conducted on this gene. Subcellular localization analysis revealed that the DoWRKY71 protein was localized in the nucleus. Moreover, the overexpression of DoWRKY71 enhanced the cold tolerance of transgenic tobacco and promoted ABA mediated stomatal closure. This study presents the first systematic analysis of the WRKY gene family in yam, offering new insights for studying WRKY transcription factors in yam. The functional study of DoWRKY71 lays theoretical foundation for further exploring the regulatory function of the DoWRKY71 gene in the growth and development related signaling pathway of yam.
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Ácido Abscísico , Dioscorea , Ácido Abscísico/farmacologia , Dioscorea/genética , Filogenia , Estresse Fisiológico/genética , Fatores de Transcrição/genéticaRESUMO
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.
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Aeromonas hydrophila , Perfilação da Expressão Gênica , Aeromonas hydrophila/fisiologia , Aeromonas hydrophila/genética , Perfilação da Expressão Gênica/veterinária , Animais , Transativadores/genética , Transativadores/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transcriptoma , Doenças dos Peixes/microbiologiaRESUMO
Atherosclerosis is a common cardiovascular disease caused by the abnormal expression of multiple factors and genes influenced by both environmental and genetic factors. The primary manifestation of atherosclerosis is plaque formation, which occurs when inflammatory cells consume excess lipids, affecting their retention and modification within the arterial intima. This triggers endothelial cell (EC) activation, immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation and migration, foam cell formation, lipid streaks, and fibrous plaque development. These processes can lead to vascular wall sclerosis, lumen stenosis, and thrombosis. Immune cells, ECs, and VSMCs in atherosclerotic plaques undergo significant metabolic changes and inflammatory responses. The interaction of cytokines and chemokines secreted by these cells leads to the onset, progression, and regression of atherosclerosis. The regulation of cell- or cytokine-based immune responses is a novel therapeutic approach for atherosclerosis. Statins are currently the primary pharmacological agents utilised for managing unstable plaques owing to their ability to enhance endothelial function, regulate VSMC proliferation and apoptosis by reducing cholesterol levels, and mitigate the expression and activity of inflammatory cytokines. In this review, we provide an overview of the metabolic changes associated with atherosclerosis, describe the effects of inflammatory responses on atherosclerotic plaques, and discuss the mechanisms through which statins contribute to plaque stabilisation. Additionally, we examine the role of statins in combination with other drugs in the management of atherosclerosis.
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Aterosclerose , Inibidores de Hidroximetilglutaril-CoA Redutases , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Aterosclerose/metabolismo , Apoptose , CitocinasRESUMO
Background: Alternative splicing (AS) is a biological process that allows genes to be translated into diverse proteins. However, aberrant AS can predispose cells to aberrations in biological mechanisms. RNA binding proteins (RBPs), closely affiliated with AS, have gained increased attention in recent years. Among these RBPs, RBM25 has been reported to participate in the cardiac pathological mechanism through regulating AS; however, the involvement of RBM25 as a splicing factor in heart failure remains unclarified. Methods: RBM25 was overexpressed in H9c2 cells to explore the target genes bound and regulated by RBM25 during heart failure. RNA sequencing (RNA-seq) was used to scrutinize the comprehensive transcriptional level before identifying AS events influenced by RBM25. Further, improved RNA immunoprecipitation sequencing (iRIP-seq) was employed to pinpoint RBM25-binding sites, and RT-qPCR was used to validate specific genes modulated by RBM25. Results: RBM25 was found to upregulate the expression of genes pertinent to the inflammatory response and viral processes, as well as to mediate the AS of genes associated with cellular apoptosis and inflammation. Overlap analysis between RNA-seq and iRIP-seq suggested that RBM25 bound to and manipulated the AS of genes associated with inflammation in H9c2 cells. Moreover, qRT-PCR confirmed Slc38a9, Csf1, and Coro6 as the binding and AS regulatory targets of RBM25. Conclusion: Our research implies that RBM25 plays a contributory role in cardiac inflammatory responses via its ability to bind to and regulate the AS of related genes. This study offers preliminary evidence of the influence of RBM25 on inflammation in H9c2 cells.
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Processamento Alternativo , Insuficiência Cardíaca , Proteínas com Motivo de Reconhecimento de RNA , Fatores de Processamento de RNA , Processamento Alternativo/genética , Insuficiência Cardíaca/genética , Inflamação/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Animais , Ratos , Fatores de Processamento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genéticaRESUMO
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 â × E. lanceolatus â), 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 â × E. lanceolatus â), 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.
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Bass , Animais , Bass/genética , Pseudomonas/genética , Imunidade Inata/genética , InflamaçãoRESUMO
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.
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Doenças dos Peixes , Animais , Virulência/genética , Pseudomonas , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
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.
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Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Sistemas de Secreção Tipo VI , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Aeromonas hydrophila/fisiologia , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Ferro/metabolismo , Infecções por Bactérias Gram-Negativas/veterináriaRESUMO
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.
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Bass , Doenças dos Peixes , Infecções por Pseudomonas , Animais , Virulência/genética , Imunidade Inata/genética , Proteínas de Bactérias/genéticaRESUMO
ß-amylase (BAM) plays an important role in plant development and response to abiotic stresses. In this study, 5 DoBAM members were identified in yam (Dioscorea opposita Thunb.). A novel ß-amylase gene BAM1, (named DoBAM1), was isolated from yam varieties Bikeqi and Dahechangyu. The open reading frame (ORF) of DoBAM1 is 2806 bp and encodes 543 amino acids. Subcellular localization analysis indicates that DoBAM1 localizes to the cell membrane and cytoplasm. In the yam variety Dahechangyu, the starch content, ß-amylase activity, and expression of DoBAM1 were characterized and found to all be higher than in Bikeqi. DoBAM1 overexpression in tobacco is shown to promote the accumulation of soluble sugar and chlorophyll content and to increase the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ß-amylase. Under cold treatment, we observed the induced upregulation of DoBAM1 and lower starch content and malondialdehyde (MDA) accumulation than in WT plants. In conclusion, these results demonstrate that DoBAM1 overexpression plays an advanced role in cold tolerance, at least in part by raising the levels of soluble sugars that are capable of acting as osmolytes or antioxidants.
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Dioscorea , beta-Amilase , Dioscorea/genética , Nicotiana/metabolismo , beta-Amilase/genética , beta-Amilase/metabolismo , Amido/genética , Carboidratos , AçúcaresRESUMO
Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker (Larimichthys crocea) and orange-spotted grouper (Epinephelus coioides). Previously, RNA sequencing showed that P. plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection. To explore the role of flgK in P. plecoglossicida pathogenicity, RNA interference (RNAi) was performed to silence the P. plecoglossicida flgK gene, and the mutant (flgK-RNAi strain) with the best silencing efficiency (89.40%) was chosen for further study. Results showed that flgK gene silencing significantly attenuated P. plecoglossicida motility, adhesion, and biofilm formation. Compared to those fish infected with the wild-type strain of P. plecoglossicida, orange-spotted grouper infected with the flgK-RNAi strain showed a 55% increase in the survival rate and a one-day delay in time of first death, with fewer pathogens in the spleen and fewer white spots on the spleen surface. RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase (MAPK) signaling pathway was related to multiple immune-related pathways. Furthermore, arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways. These findings suggest that flgK is a virulence gene of P. plecoglossicida. Furthermore, flgK appears to be involved in the regulation of motility, adhesion, and biofilm formation in P. plecoglossicida, as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P. plecoglossicida infection.
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Bass , Perciformes , Infecções por Pseudomonas , Animais , Arginina/genética , Proteínas de Bactérias/genética , Bass/genética , Bass/metabolismo , Proteínas de Peixes/genética , Glicerofosfolipídeos , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Perciformes/genética , Perciformes/metabolismo , Pseudomonas , Infecções por Pseudomonas/veterinária , Transcriptoma , Virulência/genéticaRESUMO
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.
Assuntos
Aeromonas hydrophila , Doenças dos Peixes , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Doenças dos Peixes/microbiologia , Espécies Reativas de Oxigênio/metabolismo , ZincoRESUMO
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.
Assuntos
Bass , Doenças dos Peixes , Infecções por Pseudomonas , Animais , Proteínas de Bactérias/genética , Imunidade Inata/genética , Pseudomonas , Virulência/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias , Bass , Doenças dos Peixes , Pseudomonas/genética , RNA Interferente Pequeno/genética , Animais , Proteínas de Bactérias/genética , Bass/genética , Bass/microbiologia , Doenças dos Peixes/microbiologia , Inativação Gênica , Imunidade Inata , Pseudomonas/patogenicidade , Baço/microbiologia , Transcriptoma , Virulência/genéticaRESUMO
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.
Assuntos
Bass , Doenças dos Peixes , Infecções por Pseudomonas , Animais , Proteínas de Bactérias , Bass/genética , Interações Hospedeiro-Patógeno , Pseudomonas , Infecções por Pseudomonas/veterinária , VirulênciaRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Infecções por Pseudomonas/veterinária , Pseudomonas/genética , Animais , Bass/microbiologia , Doenças dos Peixes/microbiologia , Pseudomonas/patogenicidade , Infecções por Pseudomonas/imunologia , Interferência de RNA , RNA-Seq , Transcriptoma , VirulênciaRESUMO
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.