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Streptococcus agalactiae is a Gram‐positive facultative intracellular bacterium that leads to severe economic loss of tilapia worldwide. Previous studies demonstrated that CD40 contributes to host protection against intracellular injection. In this study, CD40 was characterized from Nile tilapia (Oreochromis niloticus), named OnCD40. Sequence analysis showed that open reading frame of OnCD40 was 933 bp, containing a single peptide, a transmembrane domain and four cysteine‐rich domains. The qRT‐PCR revealed that OnCD40 was expressed in all examined tissues with the most abundant ones in spleen and thymus. After S. agalactiae stimulation, the expression of OnCD40 was significantly induced in most of the detected organs. Moreover, OnCD40‐overexpressing fish elicited significant protection against subsequent S. agalactiae challenge; approximately 10000‐fold fewer bacteria were detected in spleen of OnCD40‐overexpressing fish in comparison with control fish. Thus, CD40 had protecting function in Nile tilapia against intracellular pathogens. [ABSTRACT FROM AUTHOR]

Fish appear to harbour a complex type I IFN repertoire containing subgroups a, b, c, d, e, f, and h, and IFNh is only reported in perciform fishes. However, no multiple copies of IFNh gene has been identified in fish to date. In this study, two IFNh genes named On-IFNh1 and On-IFNh2 were cloned from Nile tilapia, Oreochromis niloticus. The predicted proteins of On-IFNh1 and On-IFNh2 contain several structural features known in type I IFNs, and estimation of divergence time revealed that these two genes may have arisen from a much recent local duplication event. On-IFNh genes were constitutively expressed in all tissues examined, with the highest expression level observed in gill, and were rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, both recombinant On-IFNh1 and On-IFNh2 trigger a relative delayed but sustained induction of interferon-stimulated genes (ISGs), whereas recombinant On-IFNc elicits a rapid and transient expression of ISGs in vivo. The present study thus contributes to a better understanding of the functional properties of tilapia interferons, and also provides a new insight into the evolution of IFNh in fish. • Multiple copies of IFNh gene were first discovered in fish. • Tilapia IFNh genes may have arisen from a recent duplication event. • Tilapia IFNh genes are significantly up-regulated following the stimulation of poly(I:C). • Tilapia IFNc elicits a rapid and transient induction of ISG expression in vivo. • Tilapia IFNh1 and IFNh2 trigger a relative delayed but sustained induction of ISG expression in vivo. [ABSTRACT FROM AUTHOR]

Neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) was a member of HECT E3 ubiquitin ligases, which participated in various biological processes. In this study, a NEDD4 was identified and analyzed in Nile tilapia, Oreochromis niloticus (OnNEDD4) and its open reading frame was 2781 bp, encoding 926 amino acids. Three conserved structure features were found in OnNEDD4, including C2 domain, WW domains and HECT domain. OnNEDD4 was constitutively expressed in all examined tissues and the highest expression level was observed in thymus. After Streptococcus agalactiae stimulation, OnNEDD4 was significantly induced in several tissues, including thymus, intestine, blood and gill. Moreover, yeast two-hybrid assay shown OnNEDD4 could interact with extracellular region of OnCD40, but this interaction didn't affect the phagocytosis of monocytes/macrophages (MO/MΦ) to S. agalactiae and A. hydrophila. Taken together, the present study suggested that OnNEDD4 participate in CD40-mediated immune response excluding phagocytosis. • OnNEDD4 homologue was cloned and characterized from Nile tilapia, Oreochromis niloticus. • OnNEDD4 was expressed in all detected tissues and up-regulated after Streptococcus agalactiae stimulation. • The interaction between OnNEDD4 and OnCD40 was verified by yeast two-hybrid assay. [ABSTRACT FROM AUTHOR]

Mammals TRAF2 played a dual role in several immune signaling transduction processes. In this study, TRAF2 was cloned from Nile tilapia, Oreochromis niloticus, which named OnTRAF2. The open reading frame was 1797 bp, encoding 598 amino acids. Amino acid alignment and phylogenetic analysis indicated that OnTRAF2 showed relatively low identify with other teleost TRAF2 proteins, with the exception of TRAF2s from Epinephelus coioides. In healthy tilapia, OnTRAF2 was expressed widely in all the examined tissues, which had highest expression level in the brain. After Streptococcus agalactiae infection, the expression level of OnTRAF2 was increased significantly at different times in several organs, implying that OnTRAF2 may be involved in host defense against S. agalactiae infection. The result of subcellular localization showed that OnTRAF2 presented in cytoplasm and nucleus of HEK293T cells. Additionally, overexpression of OnTRAF2 significantly decreased the transcriptional activity of the NF-κB reporter in HEK293T cells, yeast two-hybrid results revealed that OnTRAF2 had no interaction with E3 ubiquitin ligase OnNEDD4. These results indicated that OnTRAF2 played important function during bacterial infection, and negatively mediated the immune signaling transduction in Nile tilapia, while the mechanism need further study. 1. Tumor necrosis factor receptor-associated factor 2(OnTRAF2) homologue was cloned and characterized from Nile tilapia, Oreochromis niloticus. 2. OnTRAF2 was expressed widely in all detected tissues. After Streptococcus agalactiae stimulation, OnTRAF2 was significantly up-regulated in several tissues. 3. OnTRAF2 presented in cytoplasm and nucleus of HEK293T cells, and played a negative regulator in the activation of the NF-κB reporter. 4. OnTRAF2 had no interaction with E3 ubiquitin ligase OnNEDD4 through yeast two-hybrid assay. [ABSTRACT FROM AUTHOR]

The dsRNA-activated protein kinase R (PKR) is one of key antiviral effectors induced by interferons (IFNs), and its functions are largely unknown in tilapia, an important commercial fish species suffering from several viral infectious diseases. In the present study, a PKR gene named On-PKR was identified and cloned from Nile tilapia, Oreochromis niloticus. On-PKR gene was constitutively expressed in all tissues examined, with the highest expression level observed in head kidney and liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). Importantly, the expression of On-PKR is induced by group I and group II IFNs with distinct induction kinetics in vivo : group I IFN elicits a relative delayed but sustained induction of On-PKR, whereas group II IFN triggers a rapid and transient expression of On-PKR. Moreover, the overexpression of On-PKR has been proven to inhibit the protein translation and virus replication in fish cells. The present study thus contributes to a better understanding of the functions of antiviral effectors in tilapia, and may provide clues for the prevention and therapy of viral diseases in fish. • A PKR gene named On-PKR was identified and characterized in tilapia. • On-PKR gene is up-regulated after poly(I:C) stimulation. • On-PKR is induced by group I and group II IFNs with distinct induction kinetics. • On-PKR has the activity to inhibit protein translation in fish cells. • On-PKR has the ability to suppress virus replication in fish cells. [ABSTRACT FROM AUTHOR]

As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1β and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection. • OnCD6 was identified from Nile tilapia, Oreochromis niloticus. • OnCD6 was mainly expressed in immune tissues and induced by the challenge of S. agalactiae. • In vitro , rOnCD6E exhibited binding activities to a wide spectrum of microbes and PAMPs. • In vivo , overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival. [ABSTRACT FROM AUTHOR]

Type I interferons are a subset of cytokines playing central roles in host antiviral defense, and their effects depend on the interaction with the heterodimeric receptor complex. Surprisingly, two pairs of the receptor subunits, CRFB1 and CRFB5, and CRFB2 and CRFB5, have been identified in fish, but the studies about preferential receptor usage of different fish IFN subtypes are rather limited. In this study, the three receptor chains of type I IFNs named as On-CRFB1, On-CRFB2 and On-CRFB5 were identified in Nile tilapia, Oreochromis niloticus. These three genes were constitutively expressed in all tissues examined, with the highest expression level observed in muscle and liver, and were rapidly induced in liver following the stimulation of poly(I:C). Interestingly, it is possible that all three subtypes of tilapia IFNs are able to signal through two pairs of the receptor subunits, On-CRFB1 and On-CRFB5, and On-CRFB2 and On-CRFB5. More importantly, tilapia group I IFNs (On-IFNd and On-IFNh) preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5, and group II IFNs (On-IFNc) preferentially signal through a receptor complex comprised of On-CRFB2 and On-CRFB5. The present study thus provides new insights into the receptor usage of group I and group II IFNs in fish. • The three receptor chains of type I IFNs named as On-CRFB1/2/5 were identified in tilapia. • Tilapia CRFB1/2/5 are ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. • Tilapia group I IFNs preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5. • Tilapia group II IFNs preferentially signal through a receptor complex composed of On-CRFB2 and On-CRFB5. [ABSTRACT FROM AUTHOR]

Interferons are a family of class II α-helical cytokines playing vital roles in antiviral immune response, and little information is available to date regarding the interferon system of tilapia. In this study, a type I IFN gene, named On-IFNc, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-IFNc contains several structural features known in type I IFNs, and On-IFNc was clustered together with the known IFNc in fish into a separated clade in the phylogenetic tree. On-IFNc gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, recombinant On-IFNc has been proven to markedly induce the expression of the antiviral effectors, Mx and viperin, the signalling components, STAT1, STAT2, and IRF9, and the transcription factors, IRF3 and IRF7, as well as the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. Furthermore, recombinant On-IFNc has been proven to possess antiviral activity against ISKNV. The present study thus contributes to a better understanding of the functional properties of the type I IFN system in tilapia. • An IFNc gene named On-IFNc was identified and characterized in tilapia. • On-IFNc was ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. • Recombinant On-IFNc markedly induced the expression of the interferon-stimulated genes in fish cells. • Recombinant On-IFNc significantly induced the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. • Recombinant On-IFNc caused the significant reduction in viral burden in ISKNV-infected fish cells. [ABSTRACT FROM AUTHOR]

DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical roles in the regulation of type I IFN responses, and their functions have been well-characterized in mammals. However, little information is available regarding the function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41 contains several structural features known in DDX41, including conserved DEADc and HELICc domains, and a conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)". On-DDX41 gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver and muscle, and was inducible after poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with poly(dA:dT). The present study thus contributes to a better understanding of the functional properties of DDX41 in fish. • A DDX41 gene was identified and characterized in tilapia. • Structurally, DDX41 is highly conserved from fish to mammals. • Tilapia DDX41 was ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. • Tilapia DDX41 elicits a strong activation of fish IFN promoter with the stimulation of poly(dA:dT). [ABSTRACT FROM AUTHOR]

Nile tilapia ( Oreochromis niloticus ) is a pivotal economic fish that has been plagued by Streptococcus agalactiae infections for many years. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adaptor molecule of the interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily, which can trigger downstream signaling cascades involved in innate immunity. In this study, the full-length cDNA of TRAF6 was cloned from O. niloticus (named On-TRAF6 ), which has an open reading frame of 1716 bp, and encode a polypeptide of 571 amino acids. The predicted amino acid sequence of On-TRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. Multiple sequence alignment revealed that On-TRAF6 shares a relatively high level of identity with those of other fishes (64–98%). In healthy tilapia, mRNA expression of On-TRAF6 could be detected in all the examined tissues and the highest expression occurred in the spleen. Moreover, we found that On-TRAF6 was involved immune response of Nile tilapia following the stimulation with Streptococcus agalactiae and polyinosinic: polycytidylic acid (Poly I:C) when determined by using qPCR. The result of subcellular localization showed that On-TRAF6 distributed in the cytoplasm, and over-expression of On-TRAF6 could strongly activated NF-кB pathway in HEK293T cells. These findings indicated that On-TRAF6 may play an important role in the immune response to intracellular bacteria in Nile tilapia. [ABSTRACT FROM AUTHOR]

CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules, and its mediated adhesion plays a crucial role in different physiological and pathological phenomena, especially related to leukocyte extravasation, immune synapse stability, T cell activation and proliferation. In this study, CD166 was identified from Nile tilapia (Oreochromis niloticus , OnCD166). OnCD166 contains an open reading frame of 1671 bp that encodes a peptide of 556 amino acids, and contains five consecutive extracellular immunoglobulin domains. It's tissue distribution and expression patterns after S. agalactiae challenge were also investigated. OnCD166 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnCD166 expressions were significantly up-regulated in all tested immune tissues. Meanwhile, the recombinant OnCD166 (rOnCD166E) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnCD166E could promote phagocytosis of macrophages. Taken together, our results illustrated that OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen, which play important roles in the immune responses of Nile tilapia against bacterial pathogens. • OnCD166 was identified from Nile tilapia, Oreochromis niloticus. • OnCD166 was mainly expressed in immune tissues and induced by the challenge of S. agalactiae. • OnCD166 exhibited binding activities to a wide spectrum of microbes and PAMPs. • OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen. [ABSTRACT FROM AUTHOR]

Nk-lysin, the compound of the natural killer cells (NK) and cytotoxic T lymphocytes (CTL), is an effective antimicrobial peptide that not only presents pathogen-killing activity but also plays an important role in immunomodulatory effect. To obtain large quantity of Nk-lysin from Nile tilapia (Oreochromis niloticus) for biological assays, a 366 bp cDNA sequence encoding the mature peptide of Nk-lysin was cloned into a secretion plasmid of pPIC9K, and a large-scale of the recombinant Nk-lysin was effectively produced via Pichia pastoris expression system. Antibacterial assay shown that the recombinant Nk-lysin obviously inhibited the growth of several pathogenic bacteria, including Vibrio cholerae (MIC = 31.25 μg/mL), Pseudomonas aeruginosa (MIC = 31.25 μg/mL), and Aeromonas hydrophila (MIC = 7.81 μg/mL) which is the main pathogen of aquatic fish, including Nile tilapia. Further studies revealed that recombinant Nk-lysin significantly enhanced the phagocytic activity of monocytes/macrophages (MO/MΦ) on microspheres (0.5 and 1.0 μm beads) and bioparticles (Streptococcus agalactiae and Aeromonas hydrophila), and reactive oxygen species (ROS), these activities might be mediated by the P2X 7 receptor (P2X 7 R). Moreover, the increased expressions of pro-inflammatory factor in il - 12 and ifn - γ genes and the decreased expressions of anti-inflammatory factor in il - 10 and tgf - β genes were observed in MO/MΦ after recombinant Nk-lysin treatment significantly. Meanwhile, the expression of tlr9 , myd88 , jak1 , and nf-κb genes were up-regulated significantly in Nk-lysin-treated MO/MΦ. Taken together, these results indicate that recombinant Nk-lysin of Nile tilapia processes the capacities on antibacterial and immunomodulation, and might be a promising antibiotic substitute. Furthermore, applying P. pastoris expression system to product bioactive Nk-lysin of Nile tilapia could be sufficient to meet the requirements for both research and aquacultural application. • Recombinant Nk-lysin from Nile tilapia was effectively produced via Pichia pastoris expression system. • The recombinant inhibited obviously the growth of several pathogenic bacteria, especially Aeromonas hydrophila. • The recombinant promoted significantly the phagocytosis of monocytes and/or macrophages of fish. [ABSTRACT FROM AUTHOR]