lipoteichoic-acid and Fish-Diseases

Water temperature has a major influence on the host innate immune defence and the infectivity of pathogens in ectothermic teleosts. Toll-like receptors (TLRs) are the first and well-characterized innate immune receptors that are conserved in vertebrates. However, little is known about the effect of temperature variation on TLRs in fish species. In this study, we used adult zebrafish as a research model to investigate the effect of water temperature on TLRs. Whole genome searches identified 20 TLR homologue genes in zebrafish. Multiple sequence alignment and protein structure analysis revealed the conserved domains for these TLR proteins. To identify TLR genes related to temperature variation, TLR family genes from 12 species with different body temperatures were assigned to conduct phylogenetic analyses. Based on the phylogenetic relationships, TLR3, TLR4, TLR5 and TLR20~21 were selected as candidate genes. Immunostimulation data indicated that TLR3, TLR5, and TLR21 were more sensitive to temperature variation and their expression levels were affected in response to pathogen stimulation. Taken together, our results provide a new opportunity to understand the roles of temperature on host innate immune response in fishes and have broader implications for disease prevention in aquaculture.

Topics: Animals; Female; Fish Diseases; Immunity, Innate; Lipopolysaccharides; Male; Phylogeny; Poly I-C; Sequence Alignment; Teichoic Acids; Temperature; Toll-Like Receptors; Zebrafish

C-type lectins (CTLs) are important pathogen pattern recognition receptors that recognize carbohydrate structures. In present study, a C-type lectin domain family 4 member E-like gene from turbot, which tentatively named SmCLEC4E-like (SmCLEC4EL), was identified, and the expressional and functional analyses were performed. In our results, SmCLEC4EL showed conserved synteny with CLEC4E-like genes from several fish species in genome, and possessed a typical type II transmembrane CTL architecture: an N-terminal intracellular region, a transmembrane domain and a C-terminal extracellular region which contained a predicted carbohydrate recognition domain (CRD). In addition, SmCLEC4EL exhibited the highest expression level in spleen in healthy fish, and showed significantly induced expression in mucosal tissues, intestine and skin, under bacteria challenge. Finally, the recombinant SmCLEC4EL protein combined with LPS, PGN, LTA and five different kinds of bacteria in a dose-dependent manner, and agglutinated these bacteria strains in the presence of calcium. These findings collectively demonstrated that SmCLEC4EL, a calcium-dependent CTL, could function as a pattern recognition receptor in pathogen recognition and participate in host anti-bacteria immunity.

Topics: Amino Acid Sequence; Animals; Bacterial Infections; Fish Diseases; Fish Proteins; Flatfishes; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lectins, C-Type; Lipopolysaccharides; Peptidoglycan; Phylogeny; Sequence Alignment; Teichoic Acids

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1β-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1β, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.

Topics: Aeromonas; Animals; Cloning, Molecular; Fish Diseases; Fish Proteins; Fishes; Gram-Negative Bacterial Infections; Head Kidney; Interleukin-18; Lipopolysaccharides; Nocardia; Nocardia Infections; Spleen; Teichoic Acids

Toll-like receptors (TLRs) play important roles in the innate system by recognizing pathogen-associated molecular patterns derived from various microbes. In this study, we reported the cloning and identification of paTLR3 and paTLR4 interactor with leucine rich repeats (TRIL) cDNA from silvery pomfret (Pampus argenteus). The full-length paTLR3 and paTRIL cDNA were 2996 and 3163 bp long, respectively. Both of the two proteins contained many LRR domains, one LRR-C terminal domain and one transmembrane region, which fits with the characteristic TLR and its analogue domain architecture. Phylogenetic analyses revealed that paTLR3 and paTRIL shared the closest relationship with Lateolabrax japonicas and Notothenia coriiceps, respectively. The expression levels of paTLR3 and paTRIL varied greatly among the examined tissues with the highest expression both in liver. Following exposure to V. anguillarum flagellin, A. hydrophila lipopolysaccharide (LPS) and L. plantarum lipoteichoic acid (LTA), paTLR3 and paTRIL were all up-regulated. V. anguillarum flagellin induced the highest expression levels of paTLR3 and paTRIL. A. hydrophila flagellin and A. hydrophila LPS induced the highest expression levels of IL-1β and IL-8, respectively. The present results will provide the valuable information for understanding the structure, function and the immune defense process of paTLR3 and paTRIL in silvery pomfret.

Topics: Animals; Bacteria; Fish Diseases; Fish Proteins; Fishes; Flagellin; Gene Expression; Immunity, Innate; Intercellular Signaling Peptides and Proteins; Interleukins; Lipopolysaccharides; Membrane Proteins; Teichoic Acids; Toll-Like Receptor 3

Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.

Topics: Aeromonas; Animals; Fish Diseases; Fish Proteins; Fishes; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Leukocytes; Lipopolysaccharides; Nocardia; Nocardia Infections; Poly I-C; Receptors, Tumor Necrosis Factor; Spleen; Teichoic Acids; Tumor Necrosis Factor-alpha

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.

Topics: Acute-Phase Proteins; Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Base Sequence; Blood Proteins; Carrier Proteins; Fish Diseases; Fish Proteins; Flatfishes; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Membrane Glycoproteins; Peptidoglycan; Phylogeny; Sequence Alignment; Teichoic Acids; Vibrio Infections; Vibrio vulnificus

Topics: Amino Acid Sequence; Animals; Asialoglycoprotein Receptor; Base Sequence; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacteria; Gram-Positive Bacteria; Immunity, Innate; Lectins, C-Type; Lipopolysaccharides; Phylogeny; Sequence Alignment; Teichoic Acids; Zebrafish; Zebrafish Proteins

Lipoteichoic acid (LTA) is a major constituent of the cell wall of Gram-positive bacteria. The structure and immunomodulation of LTA vary greatly between different species. LTA from Lactobacillus plantarum has been shown to exert anti-pathogenic effects. Vibrio anguillarum is a major causative agent of vibriosis, one of the most prevalent fish diseases. The purpose of this study was to examine the effects of L. plantarum LTA on V. anguillarum growth, adhesion, and induced inflammation and apoptosis in intestinal epithelial cells of silvery pomfret (Pampus argenteus). Our results showed that L. plantarum LTA was unable to inhibit V. anguillarum growth; however, it significantly inhibited adhesion of V. anguillarum. It also showed significant inhibitory effects on EHEC-induced inflammation and apoptosis by modulating the expression of NF-κB (nuclear factor kappa B), IκB (inhibitor of NF-κB), Bcl2 (B-cell leukemia/lymphoma-2), BAX (Bcl-2-associated X protein), IL-8 (interleukin 8) and TNF-α (tumor necrosis factor-α), and via inhibition of caspase-9 and caspase-3 activation. These data extend our understanding of the beneficial effects of L. plantarum LTA, which is related to the inhibition of V. anguillarum, and suggest that L. plantarum LTA has potential as a new therapeutic agent against V. anguillarum-caused vibriosis in fish.

Topics: Animals; Apoptosis; Biological Assay; Epithelial Cells; Fish Diseases; In Vitro Techniques; Inflammation; Intestines; Lactobacillus plantarum; Lipopolysaccharides; Perciformes; Probiotics; Teichoic Acids; Vibrio; Vibrio Infections