A TFPI-1 Homolog from Sebastes schlegelii: Characterization of C-Terminal Endogenous Cleavage and Antimicrobial role of Derived Peptide.

Abstract

Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor existing in two isoforms, TFPI-1 and TFPI-2. Peptides derived from the TFPI C-terminus exhibit potent antimicrobial activity. In this study, a TFPI-1 homolog from Sebastes schlegelii (SsTFPI-1) was identified, and its sequence characteristics, immune response profile, and the antimicrobial activity of its C-terminal peptide TS20 were systematically characterized. The open reading frame of SsTFPI-1 is 867 bp in length, encoding 288 amino acids that comprise a signal peptide, three Kunitz domains, and a low-complexity region. SsTFPI-1 was ubiquitously expressed across all examined tissues, with the highest expression in blood, and its transcript levels were significantly upregulated in the spleen or head kidney following stimulation with Listonella anguillarum, Edwardsiella piscicida, or poly(I:C). Subcellular localization revealed that SsTFPI-1 predominantly localizes in the cytoplasm of EPC cells, and its C-terminus undergoes endogenous cleavage. Antimicrobial assays demonstrated that TS20 exerts pronounced antibacterial activity against Micrococcus luteus with favorable thermal stability. Mechanistically, TS20 disrupts the bacterial cell membrane, enters the cells, binds to DNA, and progressively degrades it. Furthermore, TS20 significantly inhibits SVCV infection in EPC cells by targeting both viral particles and host cells, thereby reducing viral adhesion and entry, while simultaneously downregulating the expression of type I interferon (IFN-I) related genes. In vivo experiments confirmed that TS20 significantly reduced viral loads of SVCV and RBIV-C1 in common carp and turbot, respectively. Collectively, these findings elucidate the immunological role of SsTFPI-1 in S. schlegelii and underscore the potential of TS20 as a promising antimicrobial agent.