A novel programmed cell death protein 4 negatively regulates CgIL17-5 expression in hemocytes of oyster Pacific oyster (Crassostrea gigas).

The programmed cell death protein 4 (PDCD4) is a newly defined transcriptional and translational inhibitor, which plays a key role in regulating the synthesis of inflammatory cytokines in vertebrates species. In the present study, the full-length cDNA of PDCD4 from oyster Crassostrea gigas (designed as CgPDCD4) was identified to explore its possible involvement in immune response. The open reading frame of pdcd4 gene was of 1344 bp encoding a polypeptide of 447 amino acids with two conserved MA-3 domains. The deduced amino acid sequence of CgPDCD4 shared 60.18% similarity with PDCD4 from Mizuhopecten yessoensis. The mRNA transcripts of CgPDCD4 could be detected in all the tested tissues with a higher expression level in adductor muscle and hemocytes. The mRNA expression of CgPDCD4 in hemocytes was significantly down-regulated at 3 h and 6 h (0.61-fold and 0.42-fold of that in PBS group, p < 0.01, respectively) after LPS stimulation. In hemocytes, CgPDCD4 protein was found to be mainly located in the cytoplasm. After the mRNA expression of CgPDCD4 in hemocytes was knocked down (0.40-fold of that in EGFP-RNAi group) by CgPDCD4 dsRNA (dsCgPDCD4) injection, the CgIL17-5 transcripts were up-regulated (20.11-fold of that in PBS group, p < 0.01) post LPS stimulation, which was significantly higher than that in dsEGFP-injected oysters (7.06-fold of that in PBS group, p < 0.01). Meanwhile, the nuclear translocation of CgRel (homologue of Rel/NF-κB) was significantly enhanced (about 1.36-fold of that in PBS group, p < 0.01), but it was similar as that in EGFP-RNAi group (about 1.52-fold of that in PBS group, p < 0.01) after LPS stimulation. All the results suggested that CgPDCD4 in oysters played the same role as PDCD4 of vertebrates in negatively regulating the production of interleukin in immune response, but the underpinning signal pathway was not conserved during evolution.

Piperazine ring formation by a single-module NRPS and cleavage by an α-KG-dependent nonheme iron dioxygenase in brasiliamide biosynthesis.

Brasiliamides are a class of piperazine-containing alkaloids produced by Penicillium brasilianum with a range of pharmaceutical activities. The mechanism of brasiliamide biosynthesis, including piperazine ring formation and multiple tailoring modifications, still remains unclear. In this study, the biosynthetic gene cluster of brasiliamides, brs, was identified from the marine-derived fungal strain Penicillium brasilianum WZXY-M122-9. Deletion of a histone deacetylase-encoding gene using a CRISPR/Cas9 gene editing system led to the production of a new compound, namely brasiliamide I (1). The brs-encoded single-module nonribosomal peptide synthetase (NRPS) BrsA is involved in the formation of the piperazine skeleton of brasiliamides. Full-length BrsA protein (113.6 kDa) was purified, and reconstitution of enzymatic activity in vitro confirmed that BrsA stereoselectively accepts L-phenylalanine as the substrate. Multiple deletion of tailoring genes and analysis of purified proteins in vitro enabled us to propose a brasiliamide biosynthetic pathway. In the tailoring steps, an α-ketoglutarate (KG)-dependent nonheme iron dioxygenase, BrsJ, was identified to catalyze piperazine ring cleavage during biosynthesis of brasiliamide A (2). KEY POINTS: The gene cluster encoding brasiliamide biosynthesis, brs, is identified. Deletion of a histone deacetylase-encoding gene produces brasiliamide I. BrsA catalyzes brasiliamide piperazine skeleton formation. BrsJ catalyzes piperazine ring cleavage to produce brasiliamide A. Graphical abstract.

A truncated intracellular Dicer-like molecule involves in antiviral immune recognition of oyster Crassostrea gigas.

The enzyme Dicer is best known for its role as an endoribonuclease in the small RNA pathway, playing a crucial role in recognizing viral double-stranded RNA (dsRNA) and inducing down-stream cascades to mediate anti-virus immunity. In the present study, a truncated Dicer-like gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDCL) of 530 amino acids. The CgDCL contained one N-terminal DEAD domain and a C-terminal helicase domain, but lack the conserved PAZ domain, ribonuclease domain (RIBOc) and dsRNA binding domain. The mRNA transcripts of CgDCL were detected in all the examined tissues with high expression levels in lip, gills and haemocytes, which were 62.06-fold, 48.91-fold and 47.13-fold (p < 0.05) of that in mantle, respectively. In the primarily cultured oyster haemocytes, the mRNA transcripts of CgDCL were significantly induced at 12 h after poly(I:C) stimulation, which were 4.04-fold (p < 0.05) of that in control group. The expression level of CgDCL mRNA in haemocytes was up-regulated significantly after dsRNA and recombinant interferon-like protein (rCgIFNLP) injection, which was 12.87-fold (p < 0.01) and 3.22-fold (p < 0.05) of that in control group, respectively. CgDCL proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant CgDCL protein displayed binding activity to dsRNA and poly(I:C), but no obvious dsRNA cleavage activity. These results collectively suggest that truncated CgDCL from C. gigas was able to be activated by poly(I:C), dsRNA and CgIFNLP, and functioned as an intracellular recognition molecule to bind nucleic acid of virus, indicating a potential mutual cooperation between RNAi and IFN-like system in anti-virus immunity of oysters.

D1 dopamine receptor is involved in shell formation in larvae of Pacific oyster Crassostrea gigas.

Dopamine (DA), a significant member of catecholamines, is reported to induce biomineralization of calcium carbonate vaterite microspheres via dopamine receptor (DR) in bivalves, implying the modulation of dopaminergic system on shell formation during larval development. In this research, a homologue of D1 type DR (CgD1DR-1) was identified from oyster Crassostrea gigas, whose full length cDNA was 1197 bp. It was widely expressed in various tissues of C. gigas, with the significantly higher levels in hepatopancreas, mantle, muscle and gill. During developmental stages, the mRNA transcripts of CgD1DR-1 in D-shape larvae were obviously higher (p < 0.05) than those in trochophore and umbo larvae, and CO2 exposure could inhibit the synthesis of DA and mRNA expression of CgD1DR-1. After cell transfection and DA treatment, intracellular cAMP in cells with the expression of CgD1DR-1 increased significantly (p < 0.05). Furthermore, the incubation with SCH 23390 for the blockage of CgD1DR-1 significantly restrained the expressions of six shell formation-related genes including CgTyrosinase-1, CgTyrosinase-3, CgChitinaseLP, CgAMC, CgBMP and CgBMPR in trochophore and D-shape larvae. These results jointly suggested that DA together with its receptor CgD1DR-1 might be involved in shell formation during oyster larval development from trochophore to D-shape larvae, and CO2-induced ocean acidification (OA) might influence marine bivalves by inhibiting the DA-D1DR pathway to prohibit their shell formation.