Defensins are a group of small cationic and cysteine-rich peptides that are important components of the innate immune system. However, studies on defensins in teleosts are very limited, particularly studies on defensin functions through in vivo assays. In this study, we cloned and identified one β-defensin (TroBD) the golden pompano, Trachinotus ovatus, and analyzed the functions of TroBD in both in vivo and in vitro assays. TroBD is composed of 63 amino acids and shares high sequence identities (27.27-98.41%) with known β-defensins of other teleosts. The protein has a signature motif of six conserved cysteine residues within the mature peptide. The expression of TroBD was most abundant in the head kidney and spleen and was significantly upregulated following infection by Vibrio harveyi and viral nervous necrosis virus (VNNV). Purified recombinant TroBD (rTroBD) inhibited the growth of V. harveyi, and its antimicrobial activity was influenced by salt concentration. TroBD was found to have a chemotactic effect on macrophages in vitro. The results of an in vivo study demonstrated that TroBD overexpression/knockdown in T. ovatus significantly reduced/increased bacterial colonization or viral copy numbers in tissues. Taken together, these results indicate that TroBD plays a significant role in both antibacterial and antiviral immunity and provide new avenues for protection against pathogen infection in the aquaculture industry.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%-94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.

The Lymphocyte antigen-6 (Ly-6) superfamily has been considered to play an important role in the innate immunity of mammals. The functions of Ly-6 proteins are diverse since their low sequence homology. Currently, the function of Ly-6D, a member of Ly-6 family proteins, is completely unknown in teleost. In the present study, we identified and characterized a Ly-6D homologue (named PoLy-6D) from the teleost fish Paralichthys olivaceus and examined its immune function. PoLy-6D possesses a hydrophobic signal peptide, a LU domain including a conserved "LXCXXC" motif in N-terminus and a "CCXXXXCN" motif in C-terminus. Under normal physiological condition, PoLy-6D expression distributes in all the examined tissues, the highest three tissues are successively spleen, head kidney, and blood. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, PoLy-6D expression was induced and the patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoLy-6D (rPoLy-6D) inhibited the lysis of rabbit red blood cells by serum and selectively improved bacterial survival in serum. After serum were treated by antibody of rPoLy-6D, bacteriostatic effect of serum was obviously enhanced. These results indicate the importance of PoLy-6D as a complement regulator. rPoLy-6D possessed the binding activity to multiple bacteria but did not exhibit antimicrobial activities. The interaction between rPoLy-6D and bacteria suggests that PoLy-6D is involved in host clearance of pathogens probably by serving as a receptor for pathogens. Overexpression of PoLy-6D in vivo promoted the host defense against invading E. piscicida. These findings add new insights into the regulation mechanism of the complement system in teleost and emphasize the importance of Ly-6D products for the control of pathogen infection.

Vibrio harveyi is regarded as serious pathogen for marine fishes. However, host defense mechanisms involved in V. harveyi infection remain incompletely defined. The transcription factor IFN regulatory factor 7 (IRF7) is largely associated with host defense against viral infections, and the role of IRF7 during V. harveyi infection in fish has not been well illuminated previously. In this study, IRF7 from golden pompano (Trachinotus ovatus) was characterized (TroIRF7). The TroIRF7 gene is 1323 bp, which encodes 440 amino acid residues. Multiple amino acid alignments of TroIRF7 shows 30.37%-80.18% identity with other fish IRF7s, including Epinephelus coioides (80.18%), Larimichthys crocea (79.72%), Collichthys lucidus (79.26%), Miichthys miiuy (79.26%), Channa argus (78.77%), Cynoglossus semilaevis (72.67%), and Gadus morhua (65.23%). Like other IRF7s, TroIRF7 also contains 3 conserved domains: an N-terminal DNA-binding domain (DBD), an IRF association domain (IAD), and a C-terminal serine-rich domain (SRD). In the DBD, 4-5 conserved tryptophans were observed, which is a characteristic unique to all fish IRF7 members. TroIRF7 was constitutively expressed, with high levels in gill, head kidney, spleen, skin, and intestine. V. harveyi infection-induced TroIRF7 transcripts significantly up-regulation and translocation to the nucleus. TroIRF7 overexpression promote the fish to inhibit the replication of V. harveyi. And TroIRF7 knockdown led to decreased bacterial clearance in fish tissue. Furthermore, over-expression of TroIRF7 resulted in an increased production of interferon a3 and IFN signaling molecule in the spleen, suggesting that V. harveyi activates the IRF7- IFN pathway. These results suggest that TroIRF7 is an important component of immune responses against V. harveyi infection.

Cathepsin B (CTSB) is one of the typical representatives of cysteine protease family. It has the activity of both exopeptidase and endopeptidase. It plays an important role in antigen presentation, degradation, apoptosis, inflammatory response and physiological process of many diseases. In this study, CTSB of Trachinotus ovatus (TroCTSB) was cloned, and its structure and function were analyzed. The results showed that the coding region of TroCTSB was 993 bp, encoding 330 amino acid residues. The homology analysis showed that the amino acid sequence of TroCTSB was similar to that in other teleosts and mammals (68.69%-88.48%). Under normal physiological conditions, TroCTSB was widely distributed in various tissues with the highest expression level in stomach, followed by liver, and the lowest expression level in blood. The optimal pH and temperature of purified recombinant protein rTroCTSB were 5.5 and 40 °C, respectively. The toxicity test of metal ions showed that Fe2+, Cu2+, Ca2+ and Zn2+ could all inhibit the activity of TroCTSB, with Zn2+ ranking the first. In addition, after Edwardsiella tarda infection, the expression of TroCTSB was significantly up-regulated in liver, spleen and head kidney. The overexpression of TroCTSB significantly inhibited the infection of E. tarda in golden pompano tissues, and the knockdown of TroCTSB remarkably promoted the reproduction of E. tarda in golden pompano tissues in vivo. This study suggests that TroCTSB was involved in the antibacterial immune response of T. ovatus, and provided a reference for further research in elucidating the resistance mechanism of TroCTSB.