Type F scavenger receptor expressed by endothelial cells (SREC)-II from Epinephelus coioides is a potential pathogen recognition receptor in the immune response to Vibrio parahaemolyticus infection.

Scavenger receptors play a central role in defending against infectious diseases in mammals. However, the function of SRECII remains unknown in teleost fish. In this study, type F scavenger receptor expressed by endothelial cells-II (SRECII) cDNA sequence was first identified from Epinephelus coioides, named EcSRECII, which contained an N-terminal signal peptide, eight EGF/EGF-like cysteine-rich motifs and a C-terminal low-complexity region. The gene location maps revealed that EcSRECII has the conservation of synteny among selected species. Subcellular localization showed that EcSRECII was mainly located in the cytoplasm in HEK293T cells and GS cells. In healthy E. coioides, EcSRECII mRNA was highly expressed in spleen, skin, gill, thymus and head kidney. The relative EcSRECII mRNA expression after Vibrio parahaemolyticus infection was significantly up-regulated at 12 h in spleen, head kidney and thymus, but downregulated at 1 d in skin and reduced at 3 d and 1 w in spleen. Furthermore, overexpression of EcSRECII activated NF-κB and IFN-ß signaling pathway in vitro. Taken together, these results indicated that EcSRECII could be as the potential pathogen recognition receptor for involving in bacterial infection by regulating innate immunity responses in E. coioides.

MicroRNA-182-3p negatively regulates cytokines expression by targeting TLR5M in orange-spotted grouper, Epinephelus coioides.

Toll-like receptors (TLRs) as essential pattern recognition receptors in innate immunity, can recognize pathogens and trigger immune response to eliminate invading pathogens. MicroRNAs regulates multiple biological processes by suppressing mRNA translation or resulting in mRNA degradation. MiR-182 has previously been implicated in DNA repair, disease and cancer aspects. The potential role of miR-182-3p in TLR signaling pathway against pathogens is unclear. In this study, we found that the expression of miR-182-3p was up-regulated after Vibrio parahaemolyticus flagellin stimulation in grouper spleen (GS) cells, and negatively correlated with the expression of orange-spotted grouper (Epinephelus coioides) TLR5M (EcTLR5M). Then we found that miR-182-3p could directly target EcTLR5M by using bioinformatic analysis and dual-luciferase reporter assay. Dual-luciferase reporter assay also showed that miR-182-3p down-regulated the wild-type EcTLR5M 3'UTR in luciferase activity rather than the mutant group in HEK 293T cells. We further verified the effect of miR-182-3p on the activation of Nuclear factor-κB (NF-κB) signaling pathway, and found that miR-182-3p inhibitors significantly augmented flagellin-induced NF-κB phosphorylation. Additionally, we also demonstrated that the increased expression of miR-182-3p significantly suppressed the flagellin-induced EcTLR5M, pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expression. And the endogenous miR-182-3p knockdown experiments reversely verified the regulatory effect of miR-182-3p. These results suggested that miR-182-3p post-transcriptionally controls EcTLR5M expression and thereby suppresses the expression of pro-inflammatory cytokines. This study is the first to demonstrate that miR-182-3p suppresses pro-inflammatory cytokines expression by regulating the TLR signaling pathway.

Galectin-1 is an interactive protein of selenoprotein M in the brain.

Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM) is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM' mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM' was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM' was found to be galectin-1 (Gal-1). This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM' and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.

A novel scavenger receptor (EcSRECII) as a lipopolysaccharide recognition molecule involved in regulating NF-κB activation through extracellular EGF-like cysteine-rich repeat domains with lysosomes in Epinephelus coioides.

Scavenger receptors (SRs), as multifunctional pattern recognition receptors, play an important role in innate immunity in mammals, however, their function in fish is limited. Herein, scavenger receptor F2 in Epinephelus coioides (EcSRECII) induced an innate immune response to LPS in GS cells. EcSRECII markedly enhanced LPS-induced NF-κB and IFN-ß signaling pathways, whereas knockdown of EcSRECII significantly inhibited LPS-induced NF-κB and IFN-ß promoter activation. Interestingly, only retain of epidermal growth factor (EGF)/EGF-like domain in EcSRECII resulted in a punctate cytoplasmic distribution, while the C-terminal domain exhibited a distinct cytoskeletal cytoplasmic distribution. Moreover, devoid of this EGF/EGF-like domain fragment more sharply impaired its ability to activate EcSRECII-induced NF-κB activation than deletion of the C-terminal domain region, but both domains significantly induced IFN-ß promoter activation. Full-length EcSRECII and the deletion mutant of C-terminal domain could partly colocalize with lysosomes by LPS derived from V. parahaemolyticus (V.p. LPS) in GS cells, but there was no similar distribution in the deletion mutant of EGF/EGF-like domain. This finding firstly suggested that the N-terminal EGF/EGF-like domain was necessary for the NF-κB signaling pathway to trigger resistance to vibrio infection and its functional exertion may be associated with lysosomes, thus providing insights into the regulation of resistance to vibrio infection in teleosts.

Integrative analyses of mRNA and microRNA expression profiles reveal the innate immune mechanism for the resistance to Vibrio parahaemolyticus infection in Epinephelus coioides.

Vibrio parahaemolyticus, as one of the main pathogens of marine vibriosis, has brought huge losses to aquaculture. However, the interaction mechanism between V. parahaemolyticus and Epinephelus coioides remains unclear. Moreover, there is a lack of comprehensive multi-omics analysis of the immune response of grouper spleen to V. parahaemolyticus. Herein, E. coioides was artificially injected with V. parahaemolyticus, and it was found that the mortality was 16.7% in the early stage of infection, and accompanied by obvious histopathological lesions in the spleen. Furthermore, 1586 differentially expressed genes were screened by mRNA-seq. KEGG analysis showed that genes were significantly enriched in immune-related pathways, Acute-phase immune response, Apoptosis, Complement system and Cytokine-cytokine receptor interaction. As for miRNA-seq analysis, a total of 55 significantly different miRNAs were identified. Further functional annotation analysis indicated that the target genes of differentially expressed miRNAs were enriched in three important pathways (Phosphatidylinositol signaling system, Lysosome and Focal adhesions). Through mRNA-miRNA integrated analysis, 1427 significant miRNA-mRNA pairs were obtained and "p53 signaling pathway", "Intestinal immune network for IgA production" were considered as two crucial pathways. Finally, miR-144-y, miR-497-x, novel-m0459-5p, miR-7133-y, miR-378-y, novel-m0440-5p and novel-m0084-3p may be as key miRNAs to regulate immune signaling pathways via the miRNA-mRNA interaction network. The above results suggest that the mRNA-miRNA integrated analysis not only sheds new light on the molecular mechanisms underlying the interaction between host and V. parahaemolyticus but also provides valuable and new insights into resistance to vibrio infection.

A new method for multi-site-directed mutagenesis.

A modified method for multi-site-directed mutagenesis was developed here based on polymerase chain reaction (PCR), DpnI digestion, and overlap extension. It needs only methylated plasmids obtained by Dam methyltransferase or plasmids from dam(+)Escherichia coli containing target gene. The procedure consists of PCR, DpnI digestion, overlap extension PCR, and plasmid transformation. The method was developed for multi-site-directed mutagenesis, including close proximity of mutation sites. It does not require 5'-phosphorylated primers and ligation and, thus, significantly simplifies the routine work and reduces the experimental cost for multi-site-directed mutagenesis.