RESUMEN
As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-ß (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Perciformes/genética , Perciformes/inmunología , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Filogenia , Alineación de Secuencia/veterinariaRESUMEN
The complete cDNA sequence of macrophage expressed gene (saMpeg1), a perforin-like molecule, was isolated from small abalone (Haliotis diversicolor supertexta) by homology cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA of saMpeg1 was 2781 bp, consisting of a 5'-terminal untranslated region (UTR) of 252 bp, a 3'-terminal UTR of 342 bp with a signal sequence TAA and a poly (A) tail, and an open reading frame of 2184 bp. The deduced protein (saMpeg1) was composed of 728 amino acids, and contains the cytolytic "helix-turn-helix" domain of perforin (residues 171-218), of which the alpha-helices are amphipathic as are those of perforin. A putative single transmembrane domain is located at residues 667-689, and a modified furin cleavage site (KRRRK; residues 689-693) immediately follows. The result of real time quantitative PCR showed that saMpeg1 was highly expressed at 8h and 96 h post-injection of the Gram-negative bacterium Vibrio parahaemolyticus, but there was no change after TBT exposure. The structural similarity to mammalian perforin and the different gene expression level to bacterial infection and TBT exposure suggest that saMpeg1 may play a role in the immune response against microorganisms in small abalone.