ABSTRACT
In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.
Subject(s)
Gastropoda , Hemocytes , Phagocytosis , Transcriptome , Animals , Hemocytes/immunology , Hemocytes/microbiology , Hemocytes/metabolism , Gastropoda/immunology , Gastropoda/microbiology , Gastropoda/genetics , Phagocytosis/immunology , Gene Expression Profiling , Vibrio/immunology , Vibrio/physiology , Vibrio parahaemolyticus/immunology , Vibrio parahaemolyticus/physiology , Flow CytometryABSTRACT
The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering.
Subject(s)
Antibodies, Bacterial/analysis , Enzyme-Linked Immunosorbent Assay , Escherichia coli Infections/diagnosis , Escherichia coli/immunology , Fluorescent Antibody Technique/instrumentation , Reagent Strips , Vibrio Infections/diagnosis , Vibrio parahaemolyticus/immunology , Yersinia enterocolitica/immunology , Animals , Antibody Specificity , Antigens, Bacterial/immunology , Bacterial Load , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Hybridomas , Mice, Inbred BALB C , Predictive Value of Tests , Reproducibility of Results , Vibrio Infections/immunology , Vibrio Infections/microbiology , WorkflowABSTRACT
The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.
Subject(s)
NF-kappa B/metabolism , Acute-Phase Proteins , Animals , Anti-Infective Agents , Bivalvia/immunology , Carrier Proteins , DNA, Complementary/genetics , Gene Expression Regulation , Hemocytes/metabolism , Hepatopancreas/immunology , Immunity, Innate/genetics , Lipopolysaccharides , Membrane Glycoproteins , Muramidase/metabolism , Peptidoglycan/metabolism , Phylogeny , Transcription Factor RelA , Unionidae/immunology , Vibrio parahaemolyticus/immunologyABSTRACT
Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1ß in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1ß and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1ß and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis.
Subject(s)
Flagellin , Tetraodontiformes , Vibrio parahaemolyticus , Animals , Cytokines/immunology , Fish Proteins/genetics , Flagellin/immunology , Interferon-gamma/immunology , Interleukin-1beta/immunology , NF-kappa B/genetics , Tetraodontiformes/immunology , Tetraodontiformes/microbiology , Toll-Like Receptor 5/genetics , Vibrio parahaemolyticus/immunologyABSTRACT
Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection.
Subject(s)
Dietary Supplements/analysis , Disease Resistance , Penaeidae/growth & development , Plasma/chemistry , Vibrio parahaemolyticus/immunology , Animal Feed/analysis , Animals , Body Weight , Hemocytes/metabolism , Immunity, Innate , Larva/growth & development , Larva/immunology , Larva/virology , Penaeidae/immunology , Penaeidae/virology , Phagocytes/metabolism , Spray Drying , SwineABSTRACT
Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.
Subject(s)
Brachyura , Caspase 3 , Vibrio Infections , Vibrio parahaemolyticus , Animals , Arthropod Proteins/metabolism , Brachyura/enzymology , Brachyura/immunology , Brachyura/microbiology , Caspase 3/metabolism , Phylogeny , Vibrio Infections/immunology , Vibrio Infections/veterinary , Vibrio parahaemolyticus/immunologyABSTRACT
Tumor necrosis factor superfamily member 10 (TNFSF10), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Apo-2L, is one of the important members of the TNF superfamily. It is well demonstrated that TNFSF10 preferentially induces a variety of tumor cell apoptosis, and therefore exerts an important role in tumor immune surveillance. However, the function of TNFSF10 in pathogen defense is poorly understood, especially in invertebrates. The blood clam (Tegillarca granosa), an important commercial marine bivalve, plays an important ecological role in the marine ecosystem. The identification of immune genes will provide new perspective for disease control in the blood clam (T. granosa) farming. To better understand the biological function of TNFSF10 protein, the full-length cDNA of TNFSF10 homologous gene of T. granosa (TgTNFSF10) was cloned and identified for the first time, which was found to contain 1239 base pairs and encode 254 amino acids with a molecular weight of 29.5 kDa and a conserved TNF domain in the C-terminal. Quantitative RT-PCR analysis showed that TgTNFSF10 gene was constitutively expressed in all tested tissues, with the highest expression in hemocytes. LPS, Vibrio alginolyticus and Vibrio parahaemolyticus stimulations dramatically increased the expression of TgTNFSF10 in T. granosa (11.47-fold, 3.71-fold and 8.29-fold compared with the control respectively). In vitro experiments showed that recombinant TgTNFSF10 protein strongly inhibited the proliferation of HepG2 cells. Further confocal microscopy and flow cytometry analysis showed that obvious apoptosis occurred in TgTNFSF10-treated hemocytes and HepG2 cells. To sum up, our study demonstrated that TgTNFSF10 had strong apoptosis-inducing activity, which may participate in the innate immune response of T. granosa to pathogen invasion.
Subject(s)
Arcidae/immunology , TNF-Related Apoptosis-Inducing Ligand/physiology , Animals , Apoptosis/drug effects , Aquaculture , Arcidae/microbiology , Cell Proliferation/drug effects , Gene Expression , Hemocytes/cytology , Hemocytes/drug effects , Hemocytes/metabolism , Hep G2 Cells , Humans , Lipopolysaccharides/immunology , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tissue Distribution , Vibrio parahaemolyticus/immunologyABSTRACT
Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora.
Subject(s)
Fish Diseases/metabolism , Fish Proteins/metabolism , Fishes/metabolism , Galectin 3/metabolism , Immunity, Innate , Vibrio Infections/veterinary , Vibrio/pathogenicity , Aeromonas hydrophila/immunology , Aeromonas hydrophila/pathogenicity , Animals , Fish Diseases/immunology , Fish Diseases/microbiology , Fish Proteins/genetics , Fishes/genetics , Fishes/immunology , Fishes/microbiology , Galectin 3/genetics , Gene Expression Regulation , Hemagglutination , Host-Pathogen Interactions , Mutation , Pseudomonas/immunology , Pseudomonas/pathogenicity , Vibrio/immunology , Vibrio Infections/immunology , Vibrio Infections/metabolism , Vibrio Infections/microbiology , Vibrio parahaemolyticus/immunology , Vibrio parahaemolyticus/pathogenicityABSTRACT
Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus.
Subject(s)
Adhesins, Bacterial/immunology , Bacterial Vaccines/immunology , Fimbriae Proteins/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vibrio Infections/prevention & control , Vibrio parahaemolyticus/immunology , Adhesins, Bacterial/genetics , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Biofilms/growth & development , Disease Models, Animal , Fimbriae Proteins/genetics , Foodborne Diseases/microbiology , Immunization , Mice , Mice, Inbred BALB C , Raw Foods/microbiology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Seafood/microbiology , Vibrio Infections/immunology , Vibrio parahaemolyticus/genetics , Virulence , Virulence Factors/genetics , Virulence Factors/immunologyABSTRACT
Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pirA and pirB in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei. This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei.
Subject(s)
Gene Expression Profiling , Hepatopancreas/metabolism , Penaeidae/genetics , Transcriptome , Vibrio Infections/veterinary , Vibrio parahaemolyticus/pathogenicity , Animals , Antimicrobial Cationic Peptides/genetics , Chymotrypsin/genetics , Genetic Predisposition to Disease , Hepatopancreas/immunology , Hepatopancreas/microbiology , Hepatopancreas/pathology , Necrosis , Penaeidae/immunology , Penaeidae/microbiology , Serine Endopeptidases/genetics , Serine Proteases/genetics , Vibrio Infections/genetics , Vibrio Infections/immunology , Vibrio Infections/microbiology , Vibrio parahaemolyticus/immunologyABSTRACT
Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels.
Subject(s)
Muramidase/genetics , Transcription Factors/metabolism , Unionidae/immunology , Animals , Bacteriophages/genetics , Gene Expression Regulation/immunology , Gene Knockdown Techniques , Gene Transfer, Horizontal , Transcription Factors/genetics , Unionidae/genetics , Unionidae/microbiology , Vibrio parahaemolyticus/immunologyABSTRACT
Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.
Subject(s)
Bass/immunology , Fish Diseases/immunology , Fish Proteins/metabolism , L-Amino Acid Oxidase/metabolism , Amino Acid Sequence , Animals , Bass/genetics , Bass/microbiology , Cloning, Molecular , Fish Proteins/genetics , Fish Proteins/isolation & purification , L-Amino Acid Oxidase/genetics , L-Amino Acid Oxidase/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sf9 Cells , Spodoptera , Substrate Specificity/immunology , Vibrio parahaemolyticus/immunologyABSTRACT
Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.
Subject(s)
Arthropod Proteins/immunology , Fibrinogen/immunology , Penaeidae/immunology , Recombinant Proteins/immunology , Amino Acid Sequence , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/ultrastructure , Cell Line , Fibrinogen/chemistry , Fibrinogen/genetics , Fibrinogen/ultrastructure , Immunity, Innate , Insecta , Microscopy, Electron , Penaeidae/genetics , Penaeidae/microbiology , Phylogeny , Protein Conformation, alpha-Helical , Pseudomonas aeruginosa/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Vibrio parahaemolyticus/immunologyABSTRACT
B-type allatostatins (AST-B) are neuropeptides that have important physiological roles in arthropods, they have also been identified in a number of crustacean species. Recent research on neuroendocrine-immune (NEI) regulatory system in invertebrates has exploded, it reveals that the NEI network plays an indispensable role in optimizing the immune response and maintaining homeostasis. Herein, mud crab Scylla paramamosain provides a primitive and ancient model to study crosstalk between the neuroendocrine and immune systems. In this study, qRT-PCR analysis showed that the nervous system was the main production site for Sp-AST-B mRNA in S. paramamosain, while its receptor gene (Sp-AST-BR) mRNA could be detected in all the analyzed tissues including hemocytes. This reveals that AST-B might act as a pleiotropic neuropeptide. In situ hybridization further confirmed that granular cells of hemocyte subpopulations express Sp-AST-BR. Time-course analysis revealed that bacteria-analog LPS or virus-analog Poly (I:C) challenge significantly induced Sp-AST-B expression in the thoracic ganglion, and the expression of Sp-AST-BR in hemocytes were also positively changed. Furthermore, mud crabs treated with a synthetic AST-B peptide significantly increased the mRNA levels of AST-BR, nuclear factor-κB (NF-κB) transcription factor (Dorsal and Relish), pro-inflammatory cytokine (IL-16) and immune-effector molecules, and also dramatically enhanced the nitric oxide (NO) production and phagocytic activity in hemocytes. Meanwhile dsRNA-mediated knockdown of Sp-AST-B remarkably suppressed the NO concentrations, phagocytic activity and the expression of immune related genes, resulting in markedly impaired ability of crabs to inhibit bacterial proliferation in vivo. Combined, these data demonstrate that AST-B induced innate immune in the mud crab.
Subject(s)
Arthropod Proteins/metabolism , Brachyura/immunology , Hemocytes/immunology , Immunity, Innate , Neuropeptides/metabolism , Animals , Arthropod Proteins/genetics , Brachyura/metabolism , Brachyura/microbiology , Cloning, Molecular , Disease Resistance , Gene Expression Regulation , Gene Knockdown Techniques , Hemocytes/metabolism , Neuropeptides/genetics , Phagocytosis , Poly I-C , Vibrio parahaemolyticus/immunologyABSTRACT
Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.
Subject(s)
Carrier Proteins/metabolism , Penaeidae/immunology , Vibrio parahaemolyticus/immunology , White spot syndrome virus 1/immunology , Animals , Chitin/metabolism , Hepatopancreas/immunology , Hepatopancreas/microbiology , Host-Pathogen Interactions/immunology , Penaeidae/microbiology , Up-Regulation/immunologyABSTRACT
The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection.
Subject(s)
Arthropod Proteins/metabolism , Penaeidae/immunology , Toll-Like Receptor 2/metabolism , Vibrio parahaemolyticus/immunology , White spot syndrome virus 1/immunology , Animals , Arthropod Proteins/genetics , Disease Susceptibility , Gene Knockdown Techniques , Penaeidae/metabolism , Penaeidae/microbiology , Toll-Like Receptor 2/geneticsABSTRACT
This experimental studies investigated the protective efficiencies and the potential immune mechanisms of vibrio monovalent and polyvalent autogenous formalin-inactivated whole-cell bacterins (FKC) in Gilthead sea bream (Sparus aurata) cultured in Egypt. Two months post-vaccination, the relative percentage survival (RPS) was estimated after challenge with the vaccine's homologues pathogenic strains. The survival values were 100% and 83.3% in groups immunized with monovalent V. alginolyticus or V. parahaemolyticus FKC bacterins, respectively. On the other hand, survival values were 91.75% and 75% in fish groups subjected to polyvalent (V. parahaemolyticus O11: K40 & V. alginolyticus) and (V. parahaemolyticus O3: K6 & V. alginolyticus) FKC bacterins, respectively. Overall, the tested vaccine preparations were significantly increased (P < 0.05) the agglutination antibody titer, phagocytic activity, respiratory burst activity, when compared to the non-immunized control group. The current results conclude that, autogenous Vibrio vaccines provoked a promising protection against vibriosis in Gilthead sea bream cultured in Egypt, it was superior in monovalent FKC V. alginolyticus vaccine and polyvalent FKC of V. parahaemolyticus O11: K40 with V. alginolyticus vaccine that could be useful means of prevention and control of vibriosis.
Subject(s)
Adaptive Immunity , Bacterial Vaccines/administration & dosage , Fish Diseases/prevention & control , Immunity, Innate , Sea Bream , Vibrio alginolyticus/immunology , Vibrio parahaemolyticus/immunology , Animals , Fish Diseases/immunology , Fish Diseases/microbiology , Vaccines, Combined/administration & dosage , Vibrio Infections/immunology , Vibrio Infections/microbiology , Vibrio Infections/prevention & control , Vibrio Infections/veterinaryABSTRACT
Aquatic animals are frequently threated by bacterial pathogens. The most economic and efficient protection against bacterial infection are through vaccine immunization. The various serotypes of the pathogens, such as Vibrios, hurdle the development of the vaccines, especially polyvalent vaccines. Here, we demonstrate that recombinant bacterial ghost is a good candidate for multivalent vaccine. By expressing PhiX174 gene E alone or co-expressing the gene E with two genes encoding outer membrane proteins (VP1667 and VP2369) in V. parahaemolyticus, we generated the recombinant V. parahaemolyticus ghosts VPG and rVPGs respectively. Fish immunized with either VPG or rVPG showed increased survival against the infection by either V. parahaemolyticus or V. alginolyticus, with a better protective effect by immunization with rVPG. Our furthermore studies show that rVPG stimulates stronger innate immune responses by increasing the expression of tnfα, il1ß, il6, il8 and il10 as well as that of c3b, lyz, and tlr5, the key players linking the innate and adaptive immune responses upon microbial stimulation. In summary, VPG and rVPG can protect zebrafish against the infection from at least two Vibrio species, suggesting its potential value for further aquaculture vaccines development.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Vaccines/pharmacology , Fish Diseases/prevention & control , Vibrio Infections/veterinary , Vibrio parahaemolyticus/immunology , Zebrafish , Animals , Anti-Bacterial Agents/metabolism , Bacterial Vaccines/genetics , Bacterial Vaccines/metabolism , Fish Diseases/immunology , Fish Diseases/microbiology , Vibrio Infections/immunology , Vibrio Infections/microbiology , Vibrio Infections/prevention & controlABSTRACT
Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.
Subject(s)
Bacterial Vaccines/immunology , Drug Discovery/methods , Genome, Bacterial , Vibrio Infections/drug therapy , Vibrio Infections/prevention & control , Vibrio parahaemolyticus/genetics , Vibrio parahaemolyticus/immunology , Computational Biology/methods , Drug Resistance, Multiple, Bacterial/genetics , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Escherichia coli K12/genetics , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Interaction Maps , Proteome/genetics , Proteomics/methods , Vaccines, Subunit/immunology , Vibrio Infections/microbiologyABSTRACT
When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan-gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp (p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased (p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei.