Molecular Characterization and Preliminary Functional Analysis of the NLRP3 Inflammasome in Golden Pompano (Trachinotus ovatus) in Response to Streptococcus agalactiae Infection.
Aug 2026· Fish and Shellfish Immunology· pp.
111621
· 0 citations· 37 references
Medicine
TL;DR
Five core genes associated with the NLRP3 inflammasome pathway in golden pompano showed inducible and tissue-dependent expression patterns, supporting their involvement in the antibacterial immune response, and provide a basis for understanding inflammasome-related immunity in golden pompano.
Abstract
Golden pompano (Trachinotus ovatus) is an economically important marine fish in southern China, but intensive aquaculture has increased the risk of bacterial diseases, especially streptococcosis caused by Streptococcus agalactiae. As a key component of innate immunity, the NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3 inflammasome) regulates inflammatory responses through caspase-1 (Cysteine-aspartic acid protease 1) activation and the maturation of IL-1β (Interleukin-1β) and IL-18 (Interleukin-18). In this study, five core genes associated with the NLRP3 inflammasome pathway in T. ovatus including ToNLRP3, ToASC, ToCaspase-1, ToIL-1β, and ToIL-18 were cloned and characterized. Sequence analysis showed that these genes contain conserved functional domains and share evolutionary conservation with teleost homologs. Tissue distribution analysis revealed broad expression, with relatively high levels in immune-related and mucosal tissues, including the spleen, head kidney, gill, intestine, and skin. After S. agalactiae challenge, all five genes showed inducible and tissue-dependent expression patterns, supporting their involvement in the antibacterial immune response. Fluorescence-based assays showed that ToNLRP3 was mainly localized in the cytoplasm and that ToASC interacted with ToCaspase-1, indicating the potential for inflammasome-like complex assembly. These findings provide a basis for understanding inflammasome-related immunity in golden pompano.
Streptococcus iniae is a major warm-water pathogen that cause high mortality and severe economic losses in golden pompano industry. In the present study, we performed the mRNA-miRNA integrated transcriptomic analysis of spleen of golden pompano challenged with S. iniae to explore the possible regulatory mechanism to bacterial infection. In total, we excavated 5072 DEGs, of which 2765 up-regulated and 2307 down-regulated genes. KEGG enrichment analysis indicated that the DEGs were primarily enriched in immune-related pathways, such as proteasome, cytokine-cytokine receptor interaction, p53 signaling pathway, lysosome, phagosome, Herpes simplex virus 1 infection. Additionally, a protein-protein interaction (PPI) network was constructed to extract hub genes. And the result showed that 4 hub genes, comprising cd4, il10, tnfsf2, myd88, may play vital roles in response to S. iniae infection. Furthermore, a total of 46 differentially expressed miRNAs (DEMs) were identified, containing 23 known and 23 novel DEMs. By integrating mRNA and miRNA joint analysis, we established a miRNA-mRNA regulatory network, including 12 miRNAs and 14 genes. Among them, novel-miR-357 were identified as a multi-target hub miRNA. These results provide important insights into the molecular regulatory mechanisms of immune response and inflammation processes in the defense of golden pompano against S. iniae infection.
A novel piscidin was identified in largemouth bass that contained an active peptide of 25 aa with an amphipathic helix possessing distinct hydrophobic and positively charged regions, and was significantly up-regulated in spleen from 6 h to 24 h post-lipopolysaccharide (LPS) stimulation.
Results demonstrate that LvCTL functions as an important immune recognition molecule and contributes to antibacterial defense against V. parahaemolyticus in L. vannamei.
Ning Fu, Mengxin Xing, Yuyu Wang et al.· Fishes· 0 citations
Antimicrobial peptides (AMPs) are essential components of the innate immune system, with liver-expressed antimicrobial peptide 2 (LEAP2) playing a pivotal role in fish immunity. This study investigated the antimicrobial activity and mechanisms of TroLEAP2-21, a 21-amino-acid short peptide from golden pompano (Trachinotus ovatus), against Gram-positive (Lactococcus garvieae, Staphylococcus epidermidis) and Gram-negative (Vibrio alginolyticus, Vibrio harveyi) bacteria. The predicted three-dimensional structure and helical wheel projection of TroLEAP2-21 suggested typical AMP-like physicochemical features. troleap2 expression in the liver and intestine of T. ovatus was significantly upregulated post L. garvieae or V. harveyi infection, suggesting its potential involvement in antibacterial defense. In vitro, TroLEAP2-21 exhibited antibacterial activity against the tested bacterial strains, with membrane disruption, increased membrane permeability, cytoplasmic leakage, and membrane depolarization observed after peptide treatment. Gel retardation assays further indicated species-dependent association of TroLEAP2-21 with bacterial genomic DNA. In vivo, under the tested intraperitoneal injection conditions, TroLEAP2-21 was associated with improved survival and reduced tissue damage in V. harveyi-infected T. ovatus, whereas no significant survival benefit was observed against L. garvieae. Transcriptomic analysis at 48 h post-infection showed transcriptional changes in immune-related DEGs (rsad2, mx1/mx2, il-8) and enrichment of TLR and Jak-STAT signaling pathways at the transcriptional level in peptide-treated fish. FISH showed the tissue localization of tnf-α and nf-κb transcripts and revealed treatment-associated changes in fluorescence signals, and qRT-PCR of eight immune genes supported transcriptomic results in tissues at 48 h post-infection. Collectively, these findings characterize TroLEAP2-21 as a short LEAP2-derived peptide with antibacterial and immunomodulatory activities. Its comparative advantages over other LEAP2-related peptides and its practical application potential remain to be further investigated.
Wajid Ali, Ying Chen, Kai Yan et al.· Fish and Shellfish Immunolog...· 0 citations
The RelA subunit is a core component of the nuclear factor-kappa B (NF-κB) family and plays a pivotal role in regulating immune and inflammatory responses across vertebrates and invertebrates. However, its functional role remains poorly defined in marine bivalves. In this study, a RelA homolog (RpRelA) was identified and functionally characterized in the Manila clam (Ruditapes philippinarum). The full-length cDNA of RpRelA is 2055 bp, encoding a 684-amino acid protein containing a conserved Rel homology domain (RHD) and an IPT domains. Both its tertiary structure and amino acid sequence are highly conserved across species. Phylogenetic analysis indicated that RpRelA clusters with invertebrate Rel family members. Quantitative real-time PCR (qRT-PCR) revealed that RpRelA is ubiquitously expressed in all examined tissues, with the highest expression levels detected in haemocytes and gills. Following V. parahaemolyticus challenge, both the mRNA and protein levels of RpRelA were significantly upregulated. Consistently, nuclear-cytoplasmic fractionation Western blot and immunofluorescence analyses confirmed its robust nuclear translocation upon bacterial infection. Furthermore, overexpression of RpRelA significantly upregulated pro-inflammatory genes (RpIL-17 and RpTNF) and caused severe gill inflammation, whereas its silencing suppressed their expression and alleviated the inflammatory response. Collectively, these results demonstrate that RpRelA, as a core NF-κB subunit, positively regulates the inflammatory response during V. parahaemolyticus infection in Manila clam, and provide new insights into the immune regulatory mechanism of bivalves against bacterial infection.
Hao-Dong Feng, Tian Yu, Jia-Xi Liang et al.· Fish and Shellfish Immunolog...· 0 citations
T-cell receptor (TCR) signaling is essential for adaptive immunity in vertebrates, with the tyrosine kinases Fyn, Lck, and Zap-70 playing central roles in signal transduction. In the present study, the full cDNA sequences of Fyn, Lck, and Zap-70 from golden pompano (Trachinotus ovatus) were obtained by RT-PCR and RACE-PCR and their expression patterns in normal tissues and head kidney (HK) following lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (polyI:C) stimulation and Vibrio alginolyticus infection were examined by qPCR. The open reading frames of trFyn, trLck, and trZap-70 respectively encoded 537, 513, and 609 amino acids, all containing conserved similar functional domains as their mammalian counterparts. qPCR analysis revealed that all three genes were ubiquitously expressed in examined tissues, with high expression in immune-related tissues. trFyn, trLck, and trZap-70 were significantly induced in HK after LPS, polyI:C stimulation and V. alginolyticus infection. Co-immunoprecipitation (co-IP) assay demonstrated that both trFyn and trLck could interact with trZap-70 in HEK-293T cells. These findings provide molecular basis for further study of the TCR signaling and T-cell mediated immunity in golden pompano.
You-Chuan Wei, Dongqiang Gan, Wen Qi et al.· Fish and Shellfish Immunolog...· 0 citations
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