Jul 2026· Fish and Shellfish Immunology· pp.
111586
· 0 citations· 42 references
Medicine
TL;DR
This study provides a new perspective for understanding the evolutionary conservation and species specificity of molluscan innate immune signaling pathways and offers important theoretical support for further comprehension of the molecular mechanism of scallop immune defense and promoting the green control of aquatic diseases in marine farming.
Abstract
Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an atypical member of the TRAF family, with well-established roles in modulating vertebrate innate immunity, apoptosis, and intracellular signal transduction. In contrast, its functional repertoire in invertebrates, particularly among marine mollusks, remains poorly characterized. In this study, a TRAF7 ortholog from the Zhikong scallop (Chlamys farreri) was identified and designated as CfTRAF7. The cloned CfTRAF7 cDNA comprises a 1953-bp open reading frame encoding a 650-amino-acid polypeptide. Function domain prediction confirmed that the N-terminal RING finger domain and the C-terminal WD40 repeat region exhibit significant sequence homology with TRAF7 orthologs across metazoans. Phylogenetic analysis indicated that CfTRAF7 clusters with TRAF7 homologs from other mollusks. Quantitative real-time PCR assays demonstrated that CfTRAF7 mRNA was widely expressed in all tested scallop tissues, with the highest expression in hemocytes. After stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid, and peptidoglycan, the mRNA expression of CfTRAF7 was significantly upregulated, with temporal differences in response to various pathogen-associated molecular patterns. Co-immunoprecipitation confirmed that CfTRAF7 interacted with C. farreri transforming growth factor beta-activated kinase 1 (CfTAK1). Western blot analysis revealed that overexpression of CfTRAF7 in HEK293T cells significantly enhanced the phosphorylation of mitogen-activated protein kinase (MAPK) proteins (e.g. JNK and Erk1/2), whereas RNA interference (RNAi) of CfTRAF7 suppressed MAPK phosphorylation levels in vivo following LPS challenge, but had no obvious effect on p38 phosphorylation. RNAi assays demonstrated that silencing of CfTRAF7 significantly altered the expression of several immune-related genes, including CfTAK1, CfRel, CfIL17A, CfIL17D, CfTNF-like, and CfSOD, following LPS challenge. Dual-luciferase reporter assay showed that overexpression of CfTRAF7 could activate immune-related reporter genes such as interleukin-6 (IL-6), interferon α/γ (IFNα/γ), activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), and tumor necrosis factor α (TNFα), in a dose-dependent manner. This study not only enriches the functional research on TRAF7 in invertebrates but also provides a new perspective for understanding the evolutionary conservation and species specificity of molluscan innate immune signaling pathways. Additionally, it also offers important theoretical support for further comprehension of the molecular mechanism of scallop immune defense and promoting the green control of aquatic diseases in marine farming.
SiTRAF3 is identified as a pivotal scaffold protein in the echinoid innate immune system, as it significantly drove the promoter activities of nuclear factor kappa-B, activator protein-1, signal transducer and activator of transcription 3, along with multiple inflammatory cytokines and interferon-related elements.
Xiaolong Chu, Fengchen Liu, Yingying Liu et al.· Fish and Shellfish Immunolog...· 0 citations
Toll-like receptor (TLR) family proteins act as pattern recognition receptors with pivotal roles in immune defense; however, the function of TLRs in marine mollusks requires further investigation. In this study, we identified a novel TLR gene (CfTLR-Like-1 [CfTLR-L1]) in Chlamys farreri and analyzed its immune functions. CfTLR-L1 has a coding sequence of 1,785 bp and encodes 594 amino acids. It includes typical protein domains; the amino acid sequences of the protein domains are conserved. Phylogenetic analyses demonstrated a close evolutionary relationship among proteins in the mollusk TLR subfamily. CfTLR-L1 is predominantly located on the cell membrane. CfTLR-L1 mRNA is expressed in all tissues, particularly in the hepatopancreas and mantle. Pathogenic stressors, including lipopolysaccharide, peptidoglycan, polyinosinic-polycytidylic acid, Vibrio anguillarum, Vibrio alginolyticus, Vibrio splendidus, and acute viral necrosis virus, significantly increase the levels of CfTLR-L1 mRNA transcripts. The Toll/interleukin 1 receptor (TIR) domain of CfTLR-L1 interacts with the MyD88 family proteins of scallops (including CfMyD88-1, CfMyD88-3, and CfMyD88-4), implying that multiple unique TLR signal transduction mechanisms exist in marine mollusks. CfTLR-L1-TIR interacts with CfMyD88-1-P3, which contains only the TIR structural domain. Overexpression of CfTLR-L1-TIR in HEK293T cells activated reporter genes, including IFNβ, IFNγ, and STAT3. Similarly, overexpression of CfTLR-L1 significantly affected mitogen-activated protein kinase phosphorylation, specifically of p38 and c-Jun N-terminal kinase. Our findings expand the knowledge of the role of TLR in mollusk immunity and may provide evidence for selecting disease-resistant scallops for cultivation.
Jilv Ma, Fengchen Liu, Zihao Zhang et al.· Developmental and Comparativ...· 0 citations
Src, a prototypical non-receptor tyrosine kinase and central signaling hub, is well documented to play complex, and even opposing, roles in mammalian viral infections-both restricting viral replication and being hijacked by viruses to facilitate their own life cycles. However, its role in aquatic viral infections remains largely unexplored. Here, we characterized a novel Src homolog from Epinephelus coioides, designated EcSrc, and investigated its function during Singapore grouper iridovirus (SGIV) infection. EcSrc exhibited high sequence conservation with vertebrate orthologs from mammals to teleosts. In healthy fish, EcSrc transcripts predominated in head kidney and spleen, but were markedly downregulated upon SGIV challenge. Under basal conditions, EcSrc displayed a diffuse cytoplasmic distribution without discernible organelle-specific enrichment; however, at late stages of infection, it also localized to the nucleus and virus assembly sites. Overexpression of EcSrc significantly suppressed SGIV replication, and treatment with a Src family kinase inhibitor (PP2) enhanced viral replication, together indicating that EcSrc functions as a host restriction factor. EcSrc activated NF κB signaling by promoting IκBα and p65 phosphorylation and facilitating p65 nuclear translocation, which in turn induced inflammatory cytokine gene transcription. Notably, EcSrc could interact with TRAF5, an interaction that has not been previously reported. While TRAF5 alone exerted a suppressive effect on NF-κB, EcSrc overexpression possibly counteracted this inhibition, suggesting that EcSrc may relieve the TRAF5-imposed restriction on NF-κB activation. Together, our findings indicate that EcSrc promotes NF-κB-associated antiviral responses against SGIV and identify an EcSrc-TRAF5 interaction potentially involved in teleost antiviral immunity.
Lingfeng Guan, Yi Li, Ting Yu et al.· Fish and Shellfish Immunolog...· 0 citations
Gene expression in macrophages is a highly dynamic and tightly regulated process to prevent inflammation-driven pathology, such as septic shock. Previous studies in our lab identified the putative long non-coding RNA (lncRNA) gene U90926 as highly induced in macrophages following pattern recognition receptor activation by lipopolysaccharide (LPS) and other microbial ligands. Using an LPS-induced sepsis model, we found that U90926 knockout mice exhibited greater disease severity and mortality, indicating a protective role of the gene. Surprisingly, analysis of U90929 sequence identified an 87 amino acid open reading frame (ORF), encoding a novel protein, U9-ORF, containing a functional N-terminal secretion signal. We therefore hypothesized that protective effects of U90926 in sepsis are mediated by the secretion of U9-ORF and binding to receptors on other cells.
To screen for potential receptors for U9-ORF, we developed recombinant U9-ORF-Fc fusion proteins to measure binding via flow cytometry, and for use in vitro functional assays. Transfection of HEK293 cells was used to express potential receptors for U9-ORF, and cellular adhesion assays were used as a functional readout.
Multiple sequence alignments of predicted U9-ORF homologs revealed a conserved C-terminal RGD motif, suggesting that U9-ORF could bind specific integrins. Flow cytometry assays demonstrated that U9-ORF preferentially bound cells expressing integrin αvβ3, but not αIIbβ3, in an RGD- and divalent cation-dependent manner. Plate-bound U9-ORF-Fc supported cellular adhesion and spreading of cells expressing αvβ3 integrin, in an RGD-dependent manner.
U9-ORF is a novel ligand for integrin αvβ3, which is upregulated on endothelial cells during inflammation, and thus could mediate protective effects of U90926 in sepsis. Ongoing experiments aim to determine the effects of U9-ORF on endothelial barrier integrity in the presence of inflammatory stimuli.
NIAID/NIH R03 AI190196-01; NIAID/NIH R21 AI151116-01; Department of Education CMB-GAANN Fellowship
Cellular Adhesion, Migration, and Inflammation (CAM)
T. Hogan, D. Krementsov· Journal of Immunology· 0 citations
Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a vital factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system. The structure and function of IRAK1BP1 have been well investigated in mammals, yet the piscine IRAK1BP1 is poorly understood. In the present study, the first teleost fish IRAK1BP1 from blunt snout bream, maIRAK1BP1, was identified and characterized, which was composed of a 750 bp open reading frame encoding a protein of 249 amino acids. Phylogenetic analysis indicated that maIRAK1BP1, is a novel member of IRAK1BP1 family and showed the highest similarity to that of grass carp (96.28%). Amino acid sequence analysis revealed that maIRAK1BP1 shared a conserved SIMPL domain. Spatial expression pattern analysis revealed that maIRAK1BP1 was constitutively expressed in various normal tissues including heart, spleen, head kidneys, gill, kidney, muscle, intestine and liver. The maIRAK1BP1 expression in immune-relevant tissues (liver, head kidneys and kidney) and caudal fin cells (MAF) were significantly up-regulated after LPS and bacteria injection. The result from subcellular localization analysis indicated that maIRAK1BP1 protein was localized in both the cytoplasm and nucleus of MAF cells, but predominantly existed in cytoplasm. The activity of NF-κB promoter could be inhibited by overexpression of maIRAK1BP1 in HEK-293T cells. Overexpression of maIRAK1BP1 in MAF cells inhibited dramatically the transcriptional expression level of lipopolysaccharide-induced proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). In contrast, an increased expression of these cytokines was observed when maIRAK1BP1 was knocked down via siRNA. Taken together, these results indicated that maIRAK1BP1 played an important role in mediating host innate immune response to pathogen invasion, and disclosed the involvement of maIRAK1BP1 in NF-κB-mediated production of proinflammatory cytokines, which provided foundational insights into the conserved role of IRAK1BP1 in teleost immune regulation.
Yuandong Sun, Chunfang Li, Hongyang Sun et al.· Fish and Shellfish Immunolog...· 0 citations
Findings suggest that BmRNF181 acts as a negative regulator of the silkworm immune response and dramatically increased the expression of antimicrobial peptide genes.
Jiang Liu, Ying Peng, Ming Yu et al.· Journal of Invertebrate Path...· 0 citations
