Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73–98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The V_max, K_m, and k_cat of purified (r)OnTRAP5b were found to be 0.484 μmol × min⁻¹ × mg⁻¹, 2.112 mM, and 0.27 s⁻¹ with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺, and Fe³⁺) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a cationic peptide that is integral to the innate immune system. However, its regulatory function in the immunity of fish against pathogens remains largely unexplored. This study identifies a LEAP2 homolog (AoLEAP2) in clownfish, Amphiprion ocellaris. Our phylogenetic analysis places AoLEAP2 within the fish LEAP2 cluster, closely related to Tachinotus anak LEAP2. AoLEAP2 mRNA is present across various tissues, with the highest expression in the spleen. Following Vibrio harveyi infection, the AoLEAP2 mRNA levels significantly increased in the liver, spleen, gills, and intestine. The chemically synthesized AoLEAP2 mature peptide exhibits broad-spectrum antibacterial activity without displaying toxicity to FHM cells in vitro. The intraperitoneal injection of AoLEAP2 significantly suppresses the mRNA expression of the pro-inflammatory factors IL1β and TNFα induced by V. harveyi infection, demonstrating its anti-inflammatory and immune-regulatory role.

In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.