Abstract
TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp (Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro. Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection.
Highlights
Innate immunity plays a crucial role as the first line of defense protecting both lower and higher eukaryotes against pathogenic invasion [1]
The complete sequence of the carp TIR-Domain-Containing AdapterInducing Interferon-b (TRIF) Complementary DNA (cDNA) (GenBank accession No MZ169546) was 2,667 bp, containing a 5′ untranslated terminal region (UTR) of 343 bp, a 3′ UTR of 590 bp, and an open reading frame (ORF) of 1,734 bp encoding a polypeptide of 578 amino acids (Supplementary Figure 1)
The protein structure predicted by SWISS-MODEL showed that carp TRIF exhibited the typical characteristics of an N-terminal domain, a Toll/IL-1 recruitment (TIR) domain close to its C-terminus, and a C-terminal domain (Figures 1A, B)
Summary
Innate immunity plays a crucial role as the first line of defense protecting both lower and higher eukaryotes against pathogenic invasion [1]. The mechanism of innate immunity relies on a family of proteins characterized by a highly specialized structure often termed pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [2]. Upon the recognition of microbes, TLRs immediately recruit and bind distinct Toll/IL-1 recruitment (TIR) domain-containing adaptor molecules in the cytoplasm [4]. Toll-interleukin 1 receptor domain-containing adaptor molecule (TICAM-1, named TRIF) is the third TIR domain-containing adaptor protein to be described [6]. Mammalian TRIF consists of an N-terminal proline-rich region (PRR), a highly conserved TIR domain, and a C-terminal domain that harbors a receptor-interacting protein 1 (RIP1) interaction motif (RHIM) [8]. The TIR domain of TRIF interacts with the TIR domain of TLR3 as well as the TLR4-bridging adaptor TRAM [6, 10]. The C-terminal RIP homotypic interaction motif (RHIM) is crucial for NF-kB activation and apoptosis [11,12,13]
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