Molecular Characterization, Gene Expression and Antibacterial Activity Analysis of a Novel Piscidin (Piscidin-4) of Largemouth Bass (Micropterus Salmoides)

Molecular Characterization and Preliminary Functional Analysis of the NLRP3 Inflammasome in Golden Pompano (Trachinotus ovatus) in Response to Streptococcus agalactiae Infection.

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.

Identification of novel antimicrobial peptides and histone-derived cryptides in the hepatic transcriptome of Nile tilapia (Oreochromis niloticus) infected with Streptococcus agalactiae.

Nile tilapia (Oreochromis niloticus) is a valuable freshwater fish species widely cultured globally and in China. Its aquaculture production is challenged by Streptococcus agalactiae, the primary etiological agent of streptococcosis. Antimicrobial peptides (AMPs) play a critical role in the innate immune system of fish, exhibiting defensive and inhibitory effects against a wide range of pathogens. This study aimed to identify antimicrobial peptides and histone-derived cryptides in the Nile tilapia. We combined high-throughput RNA sequencing (RNA-seq) of S. agalactiae-infected tilapia hepatic tissue with in silico alignment against specialized AMP databases (APD and DRAMP). Putative candidates were evaluated via AlphaFold 3 structural modeling and validated in vivo through quantitative real-time PCR (qPCR) over a 48-hour post-infection period. Our transcriptomic profiling identified 13 high-confidence candidates: 7 classical candidates including two conserved transcripts (NK-lysin, hepcidin), two structural variants (piscidin 2, piscidin 3), and three (LEAP-2, thymosin β4, and lysozyme C) to be experimentally validated for the first time, alongside 6 novel histone-derived cryptides (Acipensin 1, Acipensin 2, Acipensin 6, Hipposin, SpHistin, and VK10) in Nile tilapia liver. In addition, AMPs and cryptides were subjected to sequence characterization and protein structure analysis. The expression change patterns of 7 AMP candidates alongside the canonical H2A (representative of histone proteins) were examined using qPCR. Significantly, our findings highlight the structural divergence of piscidin variants and the prediction of non-classical cryptides, which coincide with the transcriptional shutdown of canonical histone H2A. While classical AMPs exhibited a coordinated time-dependent expression pattern, progressing from an early enzymatic response to late nutritional immunity, this study fundamentally expands the known teleost immunological repertoire with these newly identified AMP molecules, emphasizing the defensive role against S. agalactiae infection.

Novel Hepatopancreas-Predominant C-Type Lectin (LvCTL) Contributes to Resistance Against Vibrio parahaemolyticus in Shrimp Litopenaeus vannamei

C-type lectins (CTLs) are important pattern recognition receptors (PRRs) involved in innate immune defense in crustaceans. In this study, a novel C-type lectin, designated LvCTL, was identified and functionally characterized from Litopenaeus vannamei. Sequence analysis revealed that the open reading frame (ORF) of LvCTL is 495 bp in length and encodes a 164-amino acid polypeptide containing a typical C-type lectin-like domain but lacking the classical carbohydrate-binding motifs. Notably, two novel EPF motifs were identified in the CTLD region, suggesting a potential non-canonical ligand recognition pattern. Phylogenetic analysis revealed that LvCTL clustered with other crustacean CTLs. Tissue expression analysis revealed that LvCTL exhibited the highest transcript level in the hepatopancreas. After immune stimulation, LvCTL expression in the hepatopancreas was significantly regulated by LPS, Vibrio parahaemolyticus, Staphylococcus aureus, WSSV and Poly(I: C), indicating its involvement in antimicrobial immune responses. RNA interference assays showed that knockdown of LvCTL significantly increased the bacterial burden and reduced the survival rate of shrimp after V. parahaemolyticus infection. Furthermore, recombinant LvCTL displayed broad bacterial binding activity to Gram-negative and Gram-positive bacteria and induced calcium-dependent agglutination of V. parahaemolyticus and S. aureus. Taken together, these results demonstrate that LvCTL functions as an important immune recognition molecule and contributes to antibacterial defense against V. parahaemolyticus in L. vannamei. These findings broaden our understanding of the functional diversity of shrimp CTLs and their contributions to crustacean innate immunity.

PoGPx-7, a glutathione peroxidase homolog from teleosts, exhibits bactericidal activity and activates host immune cells against bacterial infection.

In mammals, glutathione peroxidase 7 (GPx-7) is a member of the GPx family that exhibits peroxidase activity. Its immunological functions, especially in host defense against bacterial infection, remain unexplored in lower vertebrates. In this study, we identified a GPx-7 homolog from Paralichthys olivaceus (PoGPx-7) and investigated its roles during Vibrio alginolyticus infection. PoGPx-7 possesses a conserved GSH-Px domain and carries positive net charges. PoGPx-7 was constitutively expressed in various tissues, with significant upregulation upon bacterial challenge. Recombinant PoGPx-7 (rPoGPx-7) exhibited GPx activity and bound to V. alginolyticus via interaction with lipopolysaccharide and peptidoglycan. In addition, rPoGPx-7 could directly kill bacteria by disrupting membrane integrity, leading to severe structural damage and content leakage. The bactericidal activity was modulated by protein concentration, pH, temperature, and Zn2+. Furthermore, rPoGPx-7 bound to peripheral blood leukocytes (PBLs), reduced bacterial attachment and LDH release, and protected PBLs from cell death. It also significantly enhanced phagocytosis, respiratory burst, and acid phosphatase activity of PBLs. In vivo administration showed that rPoGPx-7 reduced bacterial loads in the tissues, and improved fish survival, whereas knockdown of PoGPx-7 increased susceptibility to infection. These findings provide the first evidence that teleosts GPx-7 functions as a dual-effector molecule with direct bactericidal activity and immunomodulatory capacity, providing an immunological insight of GPx family members on resistance bacterial infection.

A novel fish interferon-derived antimicrobial peptide with rapid, broad-spectrum bactericidal activity and low toxicity for aquaculture disease control

Comparative characterization of six teleost piscidins reveals distinct antimicrobial, antibiofilm and stability profiles.

Piscidins are cationic α-helical antimicrobial peptides (AMPs) that constitute a key component of the innate immune defense of teleost fish, yet the relationship between their genomic organization, structural properties, and functional specialization remains incompletely understood. In this study, six piscidin peptides from Epinephelus akaara, Seriola dumerili, Thunnus maccoyii, Argyrosomus regius, Dicentrarchus labrax, and Epinephelus coioides were characterized through an integrated sequence-to-function approach combining comparative genomics, structural modeling, physicochemical analysis, and in vitro validation, with the aim of identifying candidates with potential for biomedical and biotechnological applications. All genes studied exhibited the conserved four-exon, three-intron architecture characteristic of teleost piscidins. Structural modeling and circular dichroism confirmed α-helical conformations under membrane-mimetic conditions, despite measurable differences in hydrophobicity, charge distribution, and predicted membrane insertion parameters. Antimicrobial assays revealed distinct functional profiles: Sd_FI25 and Epinecidin_1 displayed broad antibacterial activity against Gram-positive and Gram-negative pathogens, whereas Dl_FI22 showed selective activity with reduced temporal persistence associated with lower peptide stability. Ea_FF25 exhibited comparatively weak antibacterial potency. Antibiofilm activity varied among peptides and did not uniformly parallel planktonic MIC values. Computational predictions further suggested antiviral and antitumoral potential for several sequences, extending their prospective relevance beyond classical antibacterial roles. Conserved genomic architecture and α-helical structure coexist with pronounced functional diversification among teleost piscidins. These findings demonstrate that integrating structural prediction with experimental validation is an effective strategy for identifying fish-derived innate immune peptides as candidates for biomedical applications.