Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis.

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1ß (T. nigroviridis IL-1ß). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1ß after activation.

Cloning and characterization of chst11 from Procambarus clarkii involved in the host immune response of white spot syndrome virus and Aeromonas hydrophila.

Carbohydrate sulfotransferases 11 (chst11) is one of the enzymes that synthesize chondroitin sulfate (CS), which has extensive immune functions in vitro and plays a critical role in mediating the infection of host by pathogenic microorganisms. However, whether it has immune functions in crayfish is still poorly understood. In our previous study of transcriptome, chst11 was differentially expressed in susceptible individuals and resistant individuals of Procambarus clarkii after white spot syndrome virus (WSSV) injection. Thus, in this study, the sequence of chst11 was obtained from P. clarkii for the first time and analyzed, and the expression pattern of chst11 was investigated. Besides, the purified recombinant protein of chst11 effect in protection in WSSV infection was explored. The full length of chst11 was 1536 bp with an 831-bp open reading frame (ORF), which encoding 276 amino acids residues with a calculated molecular mass of 33.1 kDa. The chst11 contains a Sulfotransfer_2 domain, one N-glycosylation site and three O-glycosylation sites. Phylogenetic analysis results showed that chst11 had the highest similarity to Penaeus vannamei (79.93%). The expression pattern of chst11 in different tissues indicated that chst11 was expressed highest in gut, gill and hypodermis, lowest in testicular duct, periesophageal nerve and hemocytes. The chst11 had different expression patterns in different tissues when the crayfish was challenged by WSSV, Aeromonas hydrophila and CpG ODN. Recombinant chst11 protein significantly reduced the amount of WSSV copy number in hepatopancreas at 6 h and 12 h post injection compared to the control group injected with bovine serum albumin (BSA). It was found that chst11 protein enhanced the expression of peroxinectin, proPO in hepatopancreas and midgut and the C-type lectin (ctl) in hemocytes and hepatopancreas. Intramuscularly injection of juvenile crayfish with chst11 protein decreased 60% mortality compared to the control group with BSA. This study is the first report on the antiviral function of chst11 in the immune system of crustacean.