Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus).

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1ß), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin.

NK-lysin, a homologue of granulysin among human, is predominantly found in natural killer cells and cytotoxic T-lymphocytes, which plays a pivotal part in innate immune responses against diverse pathogenic bacteria. Nonetheless, in teleosts, the research on antimicrobial activity and mechanisms of NK-lysin are seldom reported. In this study, we determined the antimicrobial activity of the truncated peptide TroNKL-27 that derived from golden pompano (Trachinotus ovatus) NK-lysin, and investigated its antimicrobial mechanisms. The results showed that TroNKL-27 had considerable antimicrobial potency against both Gram-positive (Staphylococcus aureus, Streptococcus agalactiae) and Gram-negative bacteria (Vibrio harveyi, V. alginolyticus, Escherichia coli, Edwardsiella tarda). Cytoplasmic membrane depolarization and propidium iodide (PI) uptake assay manifested that TroNKL-27 could induce the bacterial membrane depolarization and change its membrane permeability, respectively. In the light of scanning electron microscopy (SEM) observation, TroNKL-27 was capable of altering morphological structures of bacteria and leading to leakage of cellular contents. Moreover, the results of gel retardation assay indicated TroNKL-27 had the ability to induce the degradation of bacterial genomic DNA. As regards in vivo assay, TroNKL-27 could reduce the replication of V. harveyi in tissues of golden pompano, protect the tissue from pathological changes. Moreover, TroNKL-27 in vivo could significantly increase the expression of the immune genes (such as IL1ß, TNFα, IFN-γ, C3 and Mx) in presence or absence of V. harveyi infection. All of these results suggest that TroNKL-27 is a novel antimicrobial peptide possessing antibacterial and immunoregulatory function in vivo and in vitro, and the observed effects of TroNKL-27 will lay a solid foundation for the development of new antimicrobial agents used in aquaculture.

Macrophage migration inhibitory factor (MIF) of golden pompano (Trachinotus ovatus) is involved in the antibacterial immune response.

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-1ß), 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.