Expression analysis of tissue factor pathway inhibitors TFPI-1 and TFPI-2 in Paralichthys olivaceus and antibacterial and anticancer activity of derived peptides

Abstract

Tissue factor pathway inhibitors (TFPI), including TFPI-1 and TFPI-2, are Kunitz-type serine protease inhibitors that mainly inhibit the blood coagulation induced by tissue factors. Previous reports on teleost proved TFPI play important roles in innate immunity. In this study, two TFPI (PoTFPI-1 and PoTFPI-2) molecules from Japanese flounder (Paralichthys olivaceus) were analyzed and characterized for their expression patterns, antibacterial and anticancer activities of the C-terminal derived peptides. Quantitative real time RT-PCR analysis shows that constitutive PoTFPI-1 expression occurred, in increasing order, in the brain, muscle, spleen, gills, head kidney, blood, intestine, heart, and liver; PoTFPI-2 was expressed, in increasing order, in the brain, gills, head kidney, muscle, intestine, spleen, liver, heart, and blood. Under the stimulation of fish pathogens, both PoTFPI-1 and PoTFPI-2 expressions increased significantly in a manner that depended on the pathogens, tissue type, and infection stage. Furthermore, C-terminal peptides TP25 and TP26, derived from PoTFPI-1 and PoTFPI-2, respectively, were synthesized and proved to be active against Micrococcus luteus (for TP25 and TP26) and Staphylococcus aureus (for TP25) via retardation effects on bacterial nucleic acids. In addition, TP25 and TP26 also displayed significant inhibitory effects on human colon cancer cell line HT-29. These results reveal that both PoTFPI-1 and PoTFPI-2 play important roles in host innate immunity. The antibacterial activity and anticancer cells function of TP25 and TP26 will add new insights into the roles of teleost TFPI.

Antimicrobial and Immunoregulatory Activities of TS40, a Derived Peptide of a TFPI-2 Homologue from Black Rockfish (Sebastes schlegelii)

“…We examined the expression profiles of SsTFPI-2 in various tissues of black rockfish under normal physiological conditions, and the result showed that SsTFPI-2 expression was distributed in all the examined tissues, with it being highly expressed in the liver and spleen. The similar tissue distribution results were reported in red drum [26], tongue sole [13] and flounder [22]. The highest expression of TFPI-2 was also found in the liver in both red drum and flounder, which may be related to the role of TFPI-2 in tissue factor pathwaymediated coagulation [27].…”

“…Increasing studies have shown that teleosts TFPI-2 participated in antimicrobial immunity. Several studies had reported that bacterial or viral infection could induce TFPI-2 expression in different tissues [13,22,26]. Similarly, we also found that the expression of SsTFPI-2 was significantly induced by infection with L. anguillarum.…”

“…In this study, we analyzed and characterized a TFPI-2 homologue, SsTFPI-2, from black rockfish, and we investigated the antimicrobial activity, action mode and immune regulation characteristics of TS40, the C-terminal derived peptide of SsTFPI-2. Structurally, there are three KDs in SsTFPI-2, which connected successively between the N-terminal and C-terminal, and these are the typical structural features of TFPI-2 homologues, from teleost to human [22,24]. Moreover, it was inferred that KD1 may be the main functional domain of TFPI-2 [25].…”

“…Subsequently, TFPI-2 C-terminal peptides of several vertebrates, including chimpanzees (Pan troglodytes), mice (Mus musculus), chickens (Gallus gallus), frogs (Xenopus tropicalis), turtles (Alligator mississippiensis) and sharks (Callorhinchus milii), were shown to have antibacterial activity against Escherichia coli and Pseudomonas aeruginosa [19] and have great potential in antimicrobial therapy. However, the study of TFPI-2 in fish is limited to zebrafish (Danio rerio) [20], red drum (Sciaenops ocellatus) [21], half-smooth tongue sole (Cynoglossus semilaevis) [12,13] and Japanese flounder (Paralichthys olivaceus) [22].…”

Knockdown and Knockout of Tissue Factor Pathway Inhibitor in Zebrafish

“…Although in Japanese flounder fish, tfpia has been shown to be expressed in multiple tissues using the reverse transcription polymerase chain reaction (RT-PCR) method, and its role in innate immunity has been established, its function in coagulation has not been evaluated. 19 In this article, we generated a thrombotic phenotype due to the deficiency of tfpia both by knockdown and by generating zebrafish tfpia deletion mutant that lacks the third Kunitz domain and the C-terminus using CRISPR/Cas9 knockout method. We found that the tfpia mRNA is degraded with no protein production, suggesting that the K3 and C-terminal domain encoding messenger RNA (mRNA) sequences are important for the stability of tfpia mRNA.…”