Listonella Anguillarum Infection Research Articles

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii)

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%–87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

Molecular identification and functional analysis of KLF2 in Plecoglossus altivelis (ayu): It's regulatory role in monocyte/macrophage activation

Monocytes/macrophages (MO/MФ) play an important role in the response to infection in Plecoglossus altivelis (ayu). However, the role of transcription factors in the function of ayu MO/MФ is poorly understood. Here, we cloned the cDNA sequence of the Kruppel-like factor 2 (PaKLF2) gene from ayu. Phylogenetic analysis indicated that PaKLF2 was closest to that of Atlantic salmon (Salmo salar). Real time quantitative PCR (RT-qPCR) revealed that the PaKLF2 mRNA level was highest in the peripheral blood mononuclear cells among all tested tissues. The mRNA expression of PaKLF2 was upregulated in the head kidney, liver, spleen, and brain after Listonella anguillarum infection. Subsequently, PaKLF2 was expressed and purified to prepare anti-PaKLF2 antibodies. After L. anguillarum challenge, the PaKLF2 mRNA and protein levels were significantly upregulated in ayu MO/MФ. Moreover, PaKLF2 knockdown in MO/MФ resulted in the enhancement of cytokine production as well as phagocytotic and bactericidal capability. Therefore, PaKLF2 may modulate the immune response in ayu by suppressing the function of MO/MФ.

Molecular characterization of a CXCL8-like protein from ayu and its effect on chemotaxis of neutrophils and monocytes/macrophages.

CXCL8, a CXC-type chemokine, plays a crucial role in acute inflammation by recruiting and mediating neutrophils and other cells. In this study, the cDNA and genomic DNA sequence of a CXCL8-like protein (PaCXCL8l) from ayu (Plecoglossus altivelis) was determined. Sequence analysis showed that PaCXCL8l represented the typical structure of animal CXCL8s. Phylogenetic tree analysis indicated that PaCXCL8l was closest to CXCL8 of Atlantic cod (Gadus morhua). Constitutive expression of PaCXCL8l was detected in all tested tissues and monocytes/macrophages, and its expression dramatically increased upon Listonella anguillarum infection. In vitro, recombinant PaCXCL8l exhibited a significant chemotactic effect on neutrophils at 0.1 μg/ml and on monocytes/macrophages at 1.0 μg/ml. In vivo, the numbers of peritoneal neutrophils and monocytes/macrophages were both up-regulated following intraperitoneal administration of recombinant PaCXCL8l. These results suggest that PaCXCL8l is crucially involved in the immune response of ayu by mediating chemotaxis of neutrophils and monocytes/macrophages.

A novel lipopolysaccharide-binding protein (LBP) gene from sweetfish Plecoglossus altivelis: Molecular characterization and its role in the immune response of monocytes/macrophages

Lipopolysaccharide-binding protein (LBP) belongs to the lipid transfer/LBP (LT–LBP) family, and plays a crucial role in the recognition of bacterial components that modulate cellular signals in phagocytic cells. Although several LBPs have been identified in teleosts, the effects of LBP homologs on teleost phagocytic cells are still obscure. Here, we report the cloning a novel full-length cDNA sequence of LBP-like protein (paLBP) gene from sweetfish, Plecoglossus altivelis. The paLBP cDNA encoded a 464 aa polypeptide, which was closest to that of rainbow smelt (Osmerus mordax). paLBP mRNA was detected mainly in the spleen, liver, and head kidney and levels dramatically increased in various tissues after Listonella anguillarum infection. In contrast to mammalian studies, paLBP mRNA could also be detected in sweetfish monocytes/macrophages. Recombinant paLBP showed LPS-binding activity and Western blot results revealed a significant increase of paLBP in the supernatant of sweetfish monocytes/macrophages challenged with L. anguillarum. Moreover, paLBP neutralization led to up-regulation of IL-1β and TNF-α mRNA as well as respiratory burst activity in sweetfish monocytes/macrophages in response to L. anguillarum or LPS challenge. Therefore, these results suggest that paLBP is an inducible acute-phase protein mediating the immune response of sweetfish monocytes/macrophages upon bacterial challenge.

Molecular cloning, characterization and expression analysis of a novel wap65-1 gene from Plecoglossus altivelis

Warm temperature acclimation associated 65-kDa protein 1 (WAP65-1) is a specific fish plasma glycoprotein that is possibly involved in various physiological or pathological processes. In this study, we obtained the cDNA and genomic DNA sequences of the Plecoglossus altivelis wap65-1 (Pawap65-1) gene. Multiple sequence alignment showed that Pawap65-1 is similar in structure to wap65-1 in fish. Phylogenetic analysis revealed that Pawap65-1 is most closely related to that of a rainbow trout. Pawap65-1 transcripts are present in various tissues and are most abundant in the liver. We expressed recombinant PaWAP65-1 in Escherichia coli and raised antiserum against it in mouse. Western blot analysis revealed that the higher molecular mass of PaWAP65-1 in blood plasma was caused by post-translational N-glycosylation. Quantitative real-time quantitative PCR (qPCR) and Western blot analysis data showed that the hepatic mRNA and blood plasma levels of PaWAP65-1 were both influenced by warm temperature acclimation and cadmium exposure, but not by Listonella anguillarum infection, hypo-osmotic, or cold temperature acclimation. In conclusion, our data reveals that PaWAP65-1 is a stress-related protein, and may play a role in fish acclimation to warm temperature and cadmium exposure.

Characterization of P2X7R and Its Function in the Macrophages of ayu, Plecoglossus altivelis

P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.

Molecular characterization of an IL-1β gene from ayu, Plecoglossus altivelis

IL-1β plays a crucial role as a prototypical proinflammatory cytokine in immune responses and has been shown to affect macrophage functions. However, the effects of putative IL-1β homologs on fish macrophages are still less known. Here, we cloned the full-length cDNA sequence of IL-1β (aIL-1β) gene from ayu, Plecoglossus altivelis. Phylogenetic analysis indicated that aIL-1β was closest to that of Atlantic salmon (Salmo salar). Real-time quantitative PCR (RT-qPCR) revealed that aIL-1β transcript was mainly expressed in spleen, head kidney and gill, and dramatically increased in various tissues after Listonella anguillarum infection. Subsequently, aIL-1β was prokaryotic expressed and purified to prepare anti-aIL-1β antibody. After L. anguillarum challenge, the aIL-1β mRNA and protein levels were significantly up-regulated in ayu monocytes/macrophages. Moreover, aIL-1β neutralization did not change phagocytic capability, but reduced bacterial killing capability in ayu head kidney-derived monocytes/macrophages. Therefore, aIL-1β may play an important role in immune response of ayu, especially, contributing to bacterial killing of monocytes/macrophages.

Molecular cloning, sequence analysis and expression of ayu complement component <I>C9</I> gene

C9, a component of the membrane attack complex, participates in the final stage of the complement cascade which lyses foreign organisms by disrupting the integrity of their cell membranes. In the present study, a full-length ayu C9 (aC9) cDNA was cloned which contains 2,125 nucleotides and encodes a protein of 592 amino acids. A signal peptide was deposited in the N-terminal 22 residues. The deduced amino acid sequence of aC9 showed 56.8% identity to the C9 of rainbow trout, and 40.9% to 53.8% identity to the C9 of other teleosts. RT-PCR analysis demonstrated that the mRNA of aC9 was expressed in the liver, spleen, intestine, gill and muscle of healthy ayu fish with the highest level in the liver. Quantitative RT-PCR analysis showed that aC9 transcripts were significantly up-regulated in the liver at 4 h post Listonella anguillarum infection, peaked at 16 h post injection. Western blotting analysis revealed that serum aC9 significantly increased in Listonella anguillarum infected ayu fish. Our results suggested that aC9 may play an important role in fish immune response of anti-bacteria.

Identification of α1-antitrypsin as a positive acute phase protein in ayu ( Plecoglossus altivelis) associated with Listonella anguillarum infection

α1-Antitrypsin (AAT) is implicated in the regulation of a variety of mammalian immune responses and was recently identified as a major serpin in blood plasma of some fish. However, AAT expression following bacterial infection in fish has not been well described. In this study, we cloned the full-length ayu ( Plecoglossus altivelis) AAT gene cDNA. It contained a 1368-bp coding region, which encodes a 19-amino acids (aa) signal peptide and a 437-aa mature AAT containing the serpin’s signature sequence ( 427LKFDRPFMMLV 437). PNGase F digestion confirmed that the higher molecular mass of the serum AAT was caused by N-glycosylation. Phylogenetic analysis indicated that ayu AAT was closest to that of green spotted pufferfish. AAT transcripts were present in a variety of tissues, with the highest level in the liver. The real-time quantitative PCR data showed that AAT transcripts dramatically increased in various ayu tissues after Listonella anguillarum infection. Western blot analysis revealed that the serum AAT protein level significantly increased in response to inflammation, but displayed no significant changes after cadmium exposure or salinity challenge. This work represents the first report that identifies AAT as a positive acute-phase protein in ayu fish associated with bacterial infection, suggesting that it might play a role in fish innate immunity.

Alteration on the expression of ayu coagulation factor X gene upon Listonella anguillarum infection

Coagulation factor X (FX) plays an important role in the immune response of mammals. In this study, the full length cDNA sequence of the ayu FX gene, 1817 bp in length excluding 3'-polyA tail, was determined for the first time. The sequence contained an open reading frame, which encoded a protein of 453 amino acids with a molecular weight of 5.07×10(4). The predicted protein had motifs typical of animal FX, and its N-terminal 24 residues were the signal peptides. Sequence comparison showed that ayu FX shared 53% amino acid sequence identity with zebrafish FX. In healthy ayu, FX mRNA was expressed mainly in the liver and weakly in the brain and gill. After Listonella anguillarum infection, liver FX transcriptions significantly increased, and peaked at 16 h post infection. The serine protease motif of ayu FX was expressed in Escherichia coli and was subsequently used for antiserum preparation. Western blotting analysis revealed that serum FX significantly increased in bacterially infected ayu fish. In conclusion, the ayu FX gene expression was significant in the progress of bacterial infection, which suggests FX's role in fish immune response.

Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu ( Plecoglossus altivelis) associated with Listonella anguillarum infection

Suppressive subtractive hybridization (SSH) was employed to identify differentially expressed genes in ayu ( Plecoglossus altivelis) associated with Listonella anguillarum infection. 800 random clones were selected from forward and reverse subtractive libraries and 787 were successfully sequenced. After assembling, 105 contigs and 414 singletons were finally obtained, some of which were immune-related genes. A real-time quantitative PCR (RT-qPCR) analysis of the expression patterns of 28 transcripts showed that the false-positive rate was approximately 7.1%. Furthermore, Wap65-2 was overexpressed in Escherichia coli, purified and used for antiserum preparation. Western blot analysis revealed that serum Wap65-2 of ayu significantly increased after bacterial infection, suggesting that it was a positive acute-phase protein (APP).

Molecular cloning of liver Wap65 cDNA in ayu (Plecoglossus altivelis) and mRNA expression changes following Listonella anguillarum infection

The teleost warm temperature acclimation related 65 kDa protein (Wap65) is a plasma glycoprotein with the potential roles in heat adaptation, heme recycling, immune response and copper metabolism. It is most homologous to the mammalian hemopexin, which is the plasma transporter of heme. A full-length cDNA clone of the Wap65 gene, 1,534 bp in size, was isolated from the fish ayu (Plecoglossus altivelis). Its deduced amino acid sequence of 439 residues had 60.4-65.4% and 38.3-47.3% identical to fish Wap65-2-type and Wap65-1-type sequences, respectively. In phylogenetic analysis, aWap65 grouped tightly with those fish Wap65-2-type sequences. In healthy control fish, the highest mRNA signal for aWap65 was from the liver, moderately high in brain and gill, and but weaker in spleen, kidney, muscle, heart and intestine. In Listonella anguillarum-infected fish, aWap65 transcripts were significantly increased in liver, while no obvious changes in other tissues at 12 hpi. However, aWap65 transcripts were significantly increased in various tissues at 24 hpi when hemolysis developing, suggesting that aWap65 might be involved in the immune response of ayu.

A study on Listonella anguillarum Infection occurred in cultured gilt-head sea bream (Sparus aurata L.).

A study on Listonella anguillarum Infection occurred in cultured gilt-head sea bream (Sparus aurata L.).

Subclinical Listonella anguillarum infection does not impair recovery of swimming performance in rainbow trout Oncorhynchus mykiss

This study examines whether injections of the commonly used bacterial-challenge pathogen Listonella anguillarum (formerly Vibrio anguillarum) negatively impact the ability of rainbow trout Oncorhynchus mykiss Walbaum to perform repeat swimming trials. Fish were given intraperitoneal injections of either a sub-lethal (10(5) colony forming units; CFUs) or a lethal (10(7) CFUs) dose of L. anguillarum, held for 48 h, and then given 2 successive ramp critical swimming speed (Ucrit) tests separated by 45 min. Compared with saline-injected control fish, the low-dose injection did not significantly impair swimming performance and recovery. Similarly, Ucrit and re-performance for fish surviving the high-dose injection were comparable to control (2 of 6 fish died after injection and before testing). In contrast, a positive control test of seawater challenge did impair recovery of swimming performance. In view of these results and common use of L. anguillarum as a challenge pathogen for toxicological studies, it seems unlikely that the consequences of pathogenesis impact the important cardiorespiratory changes associated with exercise.