Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Functional properties of ATPIF1 in the orange-spotted grouper (Epinephelus coioides) in response to viral infection.

ATP synthase inhibitory factor 1 (ATPIF1) can activate mitochondrial autophagic pathway and mediates immune response by regulating ATP synthase activity. However, the role of fish ATPIF1 on viral infection is still unknown. In this study, we identified an ATPIF1 homolog (Ec-ATPIF1) from orange-spotted grouper (Epinephelus coioides). Ec-ATPIF1 is mainly expressed in the kidney and liver. The expression of Ec-ATPIF1 was significantly up-regulated after RGNNV stimulation in vitro. Further experiments showed that overexpression of Ec-ATPIF1 inhibited the expression of viral genes (CP and RdRp) and intracellular ATP synthesis. Ec-ATPIF1 overexpression also promoted the expression of mitophagy related genes (PINK1, Parkin, BNIP3, NIX, FUNDC1, LC3), inflammation-related factors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, TLR2) and interferon pathway factors (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF). While the knockdown of Ec-ATPIF1 exhibited the opposite effects on the expression of viral genes and immune-related factors above. These data suggest that Ec-ATPIF1 can impact viral infection by regulating mitophagy, ATP synthesis, the expression of inflammatory factors and interferon pathway factors. These findings will be beneficial to better explore the immune regulatory mechanisms of fish respond to viral infection.

Roles of Hcp2, a Hallmark of T6SS2 in Motility, Adhesive Capacity, and Pathogenicity of Vibrio alginolyticus.

The type VI secretion system (T6SS) is a large secretory device, widely found in Gram-negative bacteria, which plays important roles in virulence, bacterial competition, and environmental adaptation. Vibrio alginolyticus (V. alginolyticus) is an opportunistic pathogen that causes vibriosis in aquaculture animals. V. alginolyticus possesses two type VI secretion systems (named the T6SS1 and T6SS2), but their functions remain largely unclear. In this paper, the roles of the core component of the T6SS2 cluster of V. alginolyticus HY9901, hemolysin-coregulated protein2 coding gene hcp2, are reported. Deletion of hcp2 clearly impaired the swarming motility, adhesive capacity, and pathogenicity of V. alginolyticus against zebrafish. Furthermore, transmission electron microscopy (TEM) found that the abnormal morphology of flagellum filament in the hcp2 mutant strain could be partially restored by hcp2 complementarity. By proteomic and RT-qPCR analysis, we confirmed that the expression levels of flagellar flagellin and assembly-associated proteins were remarkably decreased in an hcp2 mutant strain, compared with the wild-type strain, and could be partially restored with a supply of hcp2. Accordingly, hcp2 had a positive influence on the transcription of flagellar regulons rpoN, rpoS, and fliA; this was verified by RT-qPCR. Taken together, these results suggested that hcp2 was involved in mediating the motility, adhesion, and pathogenicity of Vibrio alginolyticus through positively impacting its flagellar system.

Effects of Chinese herbal medicines on growth performance, intestinal flora, immunity and serum metabolites of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatu♂).

The immunomodulatory roles of Chinese herbal Medicine (CHM) in aquatic animals have been well-recorded. However, how CHM impacts the intestinal microbiota and serum metabolism is not fully understood. In this study, the effects of different additive levels of CHM on the growth performance, immunity, intestinal flora and serum metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) were investigated. The addition of 0.5%, 1.0%, 1.5% and 2.0% Chinese herbal medicine compound to feed could significantly improve the weight gain rate (WGR), specific growth rate (SGR) and survival rate (SR) of grouper, reduced feed coefficient, while had no significant difference on morphometric parameter. The most significant improvement for the parameters above was observed in 1.5% group. Different addition levels of CHM could also significantly enhance the activities of ACP, AKP, SOD, CAT and LZM in serum. Accordingly, the supplementation of CHM significantly induced up-regulation of immune genes such as IL-8, IL-1ß, TNF-α, Nrf2, Lzm in the liver, spleen and head kidney of grouper, improved the resistance of grouper to V. harveyi as well. The intestinal flora analysis showed that at the phylum level, the main dominant species of intestinal microorganisms were Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, Gemmatimonadota, Desulfobacterota, Fusobacteriota and Myxococcota. At the genus level, the high abundance was Lactobacillus, Streptococcus, Bacteroides, Escherichia, Romboutsia, Sphingomonas and Muribaculaceae. The abundance of probiotics (such as Lachnospiraceae, Lactobacillaceae, Streptococcaceae, etc) in CHM-supplement groups were higher (highest in 1.5% group) compared with control group. Moreover, a total of 11 common differential metabolic pathways were screened by LC-MS metabolism analysis of serum, they were Neuroactive ligand-receptor interaction, Purine metabolism, Linoleic acid, Glycerophospholipid metabolism, Taurine and hypotaurine metabolism, Arginine and proline metabolism, ABC transporters, Aminoacyl-tRNA biosynthesis, Arachidonic acid metabolism, Drug metabolism-cytochrome P450, alpha-Linolenic acid metabolism. Also, three common differential metabolites (PI(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/22:1(13Z)), PC(22:0/20:4(5Z,8Z,11Z,14Z)) associated with intestinal health, growth and disease resistance was found. These data will contributes to a comprehensive understand for the regulatory roles of CHM on fish, which is also beneficial for the disease control and sustainable development of aquaculture.

Characterization of S100A12 from nile tilapia (Oreochromis niloticus) and its roles on inflammatory responses.

S100A12 is a member of S100 proteins family that induces pro-inflammatory response via ligating with the receptor for advanced glycation end products (RAGE) and subsequent activation of intracellular signal transduction pathways. But information about fish S100A12 remain largely unclear. In this study, the S100A12 homolog (On-S100A12) was identified from Nile tilapia (Oreochromis niloticus). On-S100A12 was mainly expressed in liver and intestine. After Streptococcus agalactiae infection in vivo, S100A12 significantly increased in brain, intestine, liver and head kidney, suggesting S100A12 might played roles in immune response. The further in vitro experiments found that recombinant protein of S100A12 (rOn-S100A12) upregulated the expression of IL1-ß, TLR2, TNF-α and inhibited the expression of IL-10, indicating On-S100A12 promoted inflammatory response and activation of M1 macrophages. The present data lay a foundation to further explore the roles of fish S100 during immune defense and will also be beneficial for better understanding of fish immune-regulatory network.

Functional characterization of BAG3 in orange-spotted grouper (Epinephelus coioides) during viral infection.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1ß and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.

Conservation of structural and interactional features of CD226 and Necl5 molecules from Nile tilapia (Oreochromis niloticus).

CD226 interacts with its ligand Necl5 as a costimulatory signal. In this study, we cloned a CD226 from Nile tilapia (Oreochromis niloticus, named OnCD226) and a Necl5 (named OnNecl5). The open reading frame of OnCD226 was 1071 bp, encoding a protein of 356 amino acids. Sequence alignment analysis indicated that OnCD226 contained two Ig-like domains in ectodomain. The open reading frame of OnNecl5 was 1155 bp, encoding a protein of 384 amino acids, and there are three lg-like domains in the extracellular domain. In healthy tilapia, OnCD226 was distributed in all tested tissues and relatively higher in the brain, while OnNecl5 was relatively higher in the skin. After Streptococcus agalactiae infection, OnCD226 has the same up-regulated expression pattern as OnNecl5 in different tissues. After HKLs stimulation with S. agalactiae and Poly I:C, respectively. OnCD226 was significantly up-regulated (0.01 < p < 0.05) at 12 h and extremely significant up-regulation was observed (p < 0.01) at 48 h and 96 h, the peak was observed at 96 h after stimulation by S. agalactiae. After stimulation by Poly I:C, OnCD226 expression was extremely significant (p < 0.01) at 72 h and 96 h, the peak was observed at 96 h. After stimulation by Keyhole limpet hemocyanin (KLH), a classical T cell-dependent antigen, the expression of OnCD226 was significantly up-regulated in blood, head kidney, spleen, and thymus. Moreover, when compared with the first challenge, the gene expression of OnCD226 which response to the second challenge was up-regulated earlier. Subcellular co-localization studies showed that OnCD226 and OnNecl5 were distributed mainly in the cytomembrane. Yeast two-hybrid results, indicated a strong interaction between OnCD226 and OnNecl5. These results suggested that OnCD226 plays an important role during pathogens infection, and the interaction between CD226 and Necl5 is conserved in Nile tilapia.

Fish natural killer enhancing factor-A (NKEF-A) enhance cytotoxicity of nonspecific cytotoxic cells against bacterial infection.

Natural killer enhancing factor (NKEF)-A/B is a member of Peroxiredoxin (Prxs) family, which is named for the function of enhancing NK cells activity. NKEF also plays essential roles in multiple physiology/pathology processes including inflammation regulation, cancer development and redox reactions. However, the regulatory effects of fish NKEF on immune cells remain largely unknown. In this study, the full-length cDNA of NKEF-A (Accession No. MK584553, designated as On-NKEF-A) was identified from Nile tilapia (Oreochromis niloticus). On-NKEF-A encoded a 198 amino acid peptide with molecular mass of 22.085 kDa. On-NKEF-A protein contained a typical 2-Cys family domain, two active sites (51aa and 172aa) that were conserved in mammals, birds, amphibians and fish. Phylogenetic analysis showed that On-NKEF-A had the closest relationship with Zebra mbuna (Maylandia zebra) NKEF. The On-NKEF-A transcription was present in all examined tissues or organs. And the relative high expression levels of On-NKEF-A was found in head kidney leucocytes and nonspecific cytotoxic cells (NCC). After Streptococcus agalactiae stimulation, On-NKEF-A was significantly up-regulated in head kidney, spleen, gill and skin. Also, On-NKEF-A was markedly induced post S. agalactiae, LPS and poly I:C stimulation in head kidney-derived NCC. Moreover, On-NKEF-A was mainly distributed in cytoplasm of fathead minnow cells (FHM cells). The further in vitro analysis found that the recombinant protein of On-NKEF-A (rOn-NKEF-A) could induce the expression of various molecular markers of B cells, macrophages and NCC, enhanced the cytotoxicity of NCC via increasing the effectors expression. The present data collectively indicate that On-NKEF-A participates in anti-bacterial immune response via regulating NCC activity, which will provide new ideas to further explore the defense mechanism of fish against bacteria.

Characterization and functional analysis of a galectin-related protein B from Nile tilapia involved in the immune response to bacterial infection.

Galectin-related protein is a kind of lectin without canonical activity that regulates cell adhesion and cell growth. In this study, a novel galectin-related protein B (OnGRPB) was identified from Nile tilapia (Oreochromis niloticus). The open reading frame of OnGRPB was 438 bp and encoded a peptide of 145 amino acids. The deduced protein sequence of OnGRPB possessed a conserved carbohydrate recognition domain (CRD) with partial sugar binding sites (N-R, V-N and W-E) and shared high identities with other fish GRPB proteins. The qRT-PCR analysis found that OnGRPB was widely distributed in various tissues and monocyte/macrophages (Mo/MΦ) of healthy tilapia. After Streptococcus agalactiae infection, OnGRPB transcripts were significantly up-regulated in liver, spleen, head kidney and Mo/MΦ. The recombinant OnGRPB protein (rOnGRPB) had the binding activity and agglutination ability to bacteria. Also, rOnGRPB could modulate antibacterial activity and inflammatory factor expression of Mo/MΦ. These data collectively indicate that OnGRPB plays roles in the immune response of Nile tilapia against bacterial infection.

Identification of BAG5 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Bcl-2-associated athanogene 5 (BAG5) is a kind of molecular chaperone that can bind to the Bcl-2 and modulate cell survival. However, little is known about the functions of fish BAG5. In this study, we characterized a BAG5 homolog from orange-spotted grouper (Epinephelus coioides) gene (Ec-BAG5) and investigated its roles during viral infection. The Ec-BAG5 protein encoded 468 amino acids with four BAG domains, which shared high identities with reported BAG5. The highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte (PBL). And the Ec-BAG5 expression were significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) or Lipopolysaccharide (LPS) stimulation in vitro. Furthermore, Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)). Also, overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor 3 (IRF3), interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 (IFP35), myxovirus resistance gene 1 (Mx1) and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), as well as the activities of NF-κB, ISRE and IFN-1. These data indicate that Ec-BAG5 can affect viral infection through regulating the expression of IFN- and inflammation-related factors, which provide useful information to better understand the immune response against viral infection.

Single-cell RNA-seq reveals different subsets of non-specific cytotoxic cells in teleost.

Non-specific cytotoxic cells (NCC) are important cytotoxic leukocytes in teleost immune system. However, the NCC subsets have not been clarified. Thus, we create a comprehensive cell map of ~24,062 head kidney-derived leukocytes from Nile tilapia post poly I:C stimulation using single-cell RNA sequencing (scRNA-seq). Based on cell heterogeneity and known markers, the cells were classified into four cell types including B cell, T cell, NCC and monocytes/macrophages (Mo/MΦ). In the meantime, the regulatory network of NCC population was predicted by WGCNA and four hub genes (Stbd1, VWF, PGP, and GRN) and one transcription factor (Hvcn1) were identified. To further study the differentiation of NCC, four subsets including memory-like NCC, mature NCC, immature NCC, and pre-NCC were revealed in NCC population for the first time. Our data will provide new insight into the biology of NCC and enable more accurate functional and developmental analysis of NCC in immune system of lower vertebrates.

A tandem-repeat galectin-4 from Nile tilapia (Oreochromis niloticus) is involved in immune response to bacterial infection via mediating pathogen recognition and opsonization.

Galectins are the family of carbohydrate-binding proteins that participate in host-pathogen interaction. In this study, a galectin-4 homolog (OnGal-4) from Nile tilapia (Oreochromis niloticus) was characterized. The open reading frame of OnGal-4 was 1194 bp, encoding a peptide of 397 amino including two CRD regions and two carbohydrate recognition sites. OnGal-4 mRNA was expressed in all examined tissues with the highest level in spleen. After Streptococcus agalactiae (S.agalactiae) challenge, the OnGal-4 expression was up-regulated in the spleen, head kidney, brain, and monocytes/macrophages (Mo/MΦ). The in vitro experiments showed that recombinant OnGal-4 (rOnGal-4) protein could bind and agglutinate S.agalactiae and A.hydrophila. Also, rOnGal-4 could induce cytokines expressions and increased bactericidal activity of Mo/MΦ. Further in vivo analysis indicated that OnGal-4 overexpression could protect O.niloticus from S.agalactiae infection through modulating inflammation response. Our study suggested that OnGal-4 could improve immune response against bacterial infection by mediating pathogen recognition and opsonization.

The in vivo roles of galectin-2 from Nile tilapia (Oreochromis niloticus) in immune response against bacterial infection.

Our previous study has recorded that the recombinant protein of Nile tilapia (Oreochromis niloticus) galectin-2 (rOnGal-2) can enhance immune response against Streptococcus agalactiae (S.agalactiae) infection in vitro. In this study, we further explored the effects of OnGal-2 in immune response against bacterial infection in vivo. The administration of rOnGal-2 could improve serum antibacterial activity (ALKP, ACP, and LZM) and antioxidant capacity (CAT, POD, and SOD). After S. agalactiae infection, rOnGal-2 injection could reduce bacterial burden and decrease tissue damage in head kidney, spleen, and liver of tilapia. Also, rOnGal-2 regulated the inflammatory-related genes expression including IL-6, IL-8 and IL-10 during bacterial infection. Furthermore, rOnGal-2 administration could increase the relative percentage survival of tilapia infected with S.agalactiae. Taken together, our results indicate that OnGal-2 can protect fish from bacterial infection through reducing bacterial burden, impairing tissue damage and modulating anti-bacterial immune response, which also can be applied as a potential vaccine adjuvant in O.niloticus culture.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

Fish Galectin8-Like Exerts Positive Regulation on Immune Response Against Bacterial Infection.

Galectin-8 is a member of the galectin family that is involved in immune response against pathogens. However, the roles of fish galectin-8 during pathogen infection require comprehensive studies. In this study, a galectin-8 homolog (OnGal8-like, OnGal8-L) was characterized from Nile tilapia (Oreochromis niloticus), and its roles in response to bacterial infection were analyzed. The OnGal8-L contains an open reading frame of 891 bp, encoding a peptide of 296 amino acids with two CRD regions of tandem-repeat galectin and two carbohydrate recognition sites. The OnGal8-L protein shares 46.42% identities with reported Oreochromis niloticus galectin-8 protein. Transcriptional expression analysis revealed that OnGal8-L was constitutively expressed in all examined tissues and was highly expressed in spleen. The transcript levels of OnGal8-L were up-regulated in the spleen, head kidney, and brain, following Streptococcus agalactiae (S. agalactiae) challenge. Further in vitro analysis indicated that the recombinant protein of OnGal8-L (rOnGal8L) could agglutinate erythrocyte, S. agalactiae, and A. hydrophila and bind S. agalactiae, A. hydrophila, and various PAMPs (lipopolysaccharides, lipoteichoic acid, poly I:C, peptidoglycan, galactose, mannose, and maltose). Also, rOnGal8L could regulate inflammatory-related gene expression, phagocytosis, and a respiratory burst of monocytes/macrophages. Moreover, in vivo analysis showed that OnGal8-L overexpression could protect O. niloticus from S. agalactiae infection through modulating serum antibacterial activity (AKP, ACP, and LZM), antioxidant capacity (CAT, POD, and SOD), and monocyte/macrophage proliferation and cytokine expression, as well as reducing bacterial burden and decreasing tissue damage. Our results collectively indicate that OnGal8-L plays important regulatory roles in immune response against bacterial infection.

Molecular characterization and expression of CD48 in Nile tilapia (Oreochromis niloticus) in response to different stimulus.

CD48 is a glycosylphosphatidylinositol-anchored protein involved in lymphocyte adhesion, activation, and costimulation. In this study, the CD48 gene of tilapia (Oreochromis niloticus, named On-CD48), was cloned from the head kidney of tilapia. The coding sequences is 654 bp and encoding 217 amino acids. The deduced amino acid sequence of On-CD48 with an estimated molecular weight of 24.4 kDa and a theoretical pI of 5.03. Amino acid alignment indicated that it had two immunoglobulin-like domain conserved region. In healthy tilapia, the On-CD48 could be detected in all the examined tissues and the highest expression level in the spleen. The expression of On-CD48 in the spleen and head kidney was decreased after immunized by formalin-inactivated Streptococcus agalactiae, and the peak was observed in the spleen at 24 h and appeared again at 96 h, and in the head kidney gradual decline before 48 h then gradually increased to the original level. qPCR analysis of inactivated S. agalactiae, LPS and Poly I:C stimulated at the whole lymphocyte level showed that the stimulation of the Poly I:C was more sensitive. Prokaryotic expression results showed that efficient expression of On-CD48 protein could be realized after induced with 0.5 mmol L-1 IPTG in E. coli BL21 (DE3) for 10 h at 18 °C. The result of subcellular localization showed that On-CD48 were evenly distributed in the whole cell of HEK-293T. Western Blot confirmed that the molecular weight of the recombinant On-CD48 was about 21 kDa, consistent with the predicted result. The results of this study will lay a strong foundation for the further study of On-CD48 molecular function in tilapia.

Construction and evaluation of Vibrio alginolyticus ΔclpP mutant, as a safe live attenuated vibriosis vaccine.

Vibrio alginolyticus is a common and serious pathogen threatening the progress of coastal aquaculture. ClpP protease has been proved to be closely associated with biofilm formation, stress tolerance, autolysis and virulence in several pathogens. Hence, targeting ClpP may be a potentially viable, attractive option for the preparation of vaccine in preventing vibriosis. In this study, an in-frame deleted mutant strain (ΔclpP) was constructed by allelic exchange mutagenesis to investigate physiological role of clpP in pathogenicity of V. alginolyticus and evaluate its potential as a live attenuated vaccine. The results exhibited that ΔclpP showed no differences in external morphology, growth, swarming motility and ECPase activity. However, ΔclpP represented an increment in biofilm formation, and a decrement in adherence to CIK cells. In addition, virulence of ΔclpP was examined in pearl gentian grouper and was found to be seriously attenuated. ΔclpP induced high antibody titers and provided a valid protection with a relative percent survival value of 83.8% without histopathologic abnormality. Our results indicated ΔclpP showed a great potential to be a live attenuated vaccine.

An IL-6 gene in humphead snapper (Lutjanus sanguineus): Identification, expression analysis and its adjuvant effects on Vibrio harveyi OmpW DNA vaccine.

Interleukin 6 (IL-6) is a pleiotropic cytokine that plays important role in mediating the innate and adaptive immune responses against pathogen infection. In this study, an IL-6 homolog (Ls-IL6) was identified and characterized from humphead snapper, Lutjanus sanguineus. The full-length cDNA of Ls-IL6 was 1066 bp, containing an open reading frame (ORF) of 639 bp encoding 212 amino acids, 5' untranslated region(UTR) of 63 bp and 3' UTR of 605 bp. The predicted Ls-IL6 protein had typical motif of IL-6 family and shared high identities to teleost IL-6s. Ls-IL6 extensively expressed in various tissues, and the highest expression of Ls-IL6 was detected in head kidney, spleen and thymus. In vivo, the transcript levels of Ls-IL6 were significantly up-regulated in response to Vibrio harveyi infection. Moreover, the DNA plasmid containing the OmpW of V. harveyi together with the gene encoding Ls-IL6 were successfully constructed and administered to fish, the protective efficacy of Ls-IL6 was investigated. Compared with the pcDNA-OmpW group, the level of specific antibodies against V. harveyi increased in pcDNA-IL6-OmpW injected group. After V. harveyi infection, the pcDNA-IL6-OmpW vaccinated fish showed higher relative percent survival (76%) than the relative survival of fish immunized with pcDNA-OmpW (60%). These results indicated that Ls-IL6 was involved in immune response against V. harveyi infection and could be applied as a promising adjuvant for DNA vaccines against V. harveyi.

Functional characterization of galectin-3 from Nile tilapia (Oreochromis niloticus) and its regulatory role on monocytes/macrophages.

Galectin-3 is a kind of ß-galactoside-binding lectin involved in host defense against pathogen infection. However, the immune functions of fish galectin-3 remain poorly understood. In this study, the roles of a fish galectin-3 (OnGal-3) from Nile tilapia (Oreochromis niloticus) on the binding activity on bacterial pathogens or PAMPs, the agglutinating activity on bacterial pathogens and the regulatory effects on monocytes/macrophages activity were investigated. After in vitro challenge of Streptococcus agalactiae and Aeromonas hydrophila, OnGal-3 expressions were significantly up-regulated in monocytes/macrophages. In addition, recombinant OnGal-3(rOnGal-3) protein showed strong binding activity on bacterial pathogens or PAMPs. Also, rOnGal-3 agglutinated Gram-positive and Gram-negative bacteria. Moreover, rOnGal-3 could induce the inflammatory factors expressions in monocytes/macrophages and enhance phagocytosis and respiratory burst activity of monocytes/macrophages. These results suggest that fish galectin-3 participates in anti-bacterial immune response through recognizing pathogens and modulating monocytes/macrophages activity.

Characterization of a tandem-repeat galectin-9 from Nile tilapia (Oreochromis niloticus) involved in the immune response against bacterial infection.

Galectin-9 is a -galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, a galectin-9 homologue (OnGal-9) was identified from Nile tilapia (Oreochromis niloticus) and its expression model and biological effects on bacterial infection were analyzed. The open reading frame of OnGal-9 sequence was 975 bp encoding 324 amino acids. It shares 45%-92% identities with other galectin-9 proteins. The deduced mature peptide of OnGal-9 possesses two conserved carbohydrate recognition domain (CRD) that connected with a linker peptide. Expression analysis indicated that OnGal-9 was distributed in all the tested tissues of healthy tilapia. The OnGal-9 expression was significantly up-regulated in spleen, head kidney, and intestine after challenged by Streptococcus agalactiae. Meanwhile, the recombinant OnGal-9 (rOnGal-9) protein displayed strong binding and agglutination activity toward both Streptococcus agalactiae and Aeromonas hydrophila. Moreover, rOnGal-9 could promote phagocytosis of macrophages. Taken together, the results here indicate that OnGal-9 might be involved in the immune response of Nile tilapia against bacterial infection.