Bid (BH3-interacting domain death agonist), a BH3-only protein of the B-cell lymphoma (BCL-2) family, has a pro-apoptotic function linking the extrinsic and intrinsic apoptotic pathways (Rigo et al., 2019). Following death receptor signaling, cytoplasmic resident Bid is cleaved by activated caspase-8 to produce a truncated form of Bid known as tBid (Li et al., 1998). tBid translocates into mitochondria and triggers cytochrome releaseCand activation of downstream caspases culminating in cell apoptosis (Wei et al., 2000). Bid has been shown to indirectly activate BAX/BAK effector proteins, neutralizing anti-apoptotic BCL-2 proteins, thus allowing BAX/BAK unhindered, spontaneous activation in the environment of the mitochondrial outer membrane, leading to the initiation of apoptosis (Huang et al., 2019 .) Bid contains a conserved BH3 domain that is required for interaction with Bcl-2 family proteins and for pro-death activity (Yin, 2006).
Bid has been reported to play a functional role in apoptosis induced by viral infection in mammals. Hepatitis C virus (HCV) core protein modulates apoptosis by enhancing Bid cleavage and activating the mitochondrial signaling pathway (Chou et al., 2005). A modified Bid containing a specific cleavage site recognized by the NS3/NS4A serine protease reduces HCV infection in mice and lowers serum HCV titers (Hsu et al., 2003). Hepatitis B virus (HBV) infection causes subsequent overexpression of death-associated receptors, which increases Bid cleavage ( Lin and Zhang, 2017 ). In epithelial cells, HEK293 Bid was required for reovirus-induced apoptosis and virus replication (Danthi et al., 2010a). Bid cleavage also occurred during influenza A virus-induced apoptosis (Yeganeh et al., 2018). The Orf virus-encoded ORFV119 protein can induce Bid expression and activation to achieve cell death (Li et al., 2018). In carp and rare minnows, Bid-deficient fish reduce carp reovirus (GCRV)-induced apoptosis (He et al., 2017). However, the function of Bida during viral infection in amphibians still remains unclear.
Chinese giant salamander iridovirus (GSIV) belongs to the large dsDNA virus family Iridoviridae, which exhibits icosahedral symmetry with a diameter of approximately 120–200 nm (Geng et al., 2011). It is the main pathogen of the Chinese giant salamander and causes severe diseases in this endangered species (Ma et al., 2014). For now, there is no effective way to prevent and treat this disease, which is the biggest threat to the survival of this species. In our previous study, GSIV infection induced typical apoptotic cell death via extrinsic and intrinsic pathways (Li et al., 2019). Moreover, Bid mRNA expression was significantly increased during the late infection process. Further studies are needed to determine the functional role of AdBid in GSIV-induced apoptosis.
This study investigated the sequence characterization and expression patterns of the Bid gene and its function in GSIV-induced apoptotic cell death using a Chinese giant salamander cell line (GSM cells). The results showed that Bid can enhance GSIV-induced apoptosis and contribute to virus replication. Together, our findings suggest that Bid promotes GSIV-induced apoptosis in the Chinese giant salamander, providing new insight into a potential regulatory mechanism during iridovirus infection.
Excerpts of Sections
Animals, cells and viruses
All animal handling procedures and experiments were approved by the Animal Care and Use Committee of the Yangtze River Fisheries Research Institute of the Chinese Academy of Fisheries Sciences. Chinese giant salamanders with an average weight of 200 g were obtained from the breeding of the Yangtze River Fisheries Research Institute. Before the experiment, the animals were kept in plastic aquariums at 20-22°C and fed diced fathead minnow meat daily for two weeks. The giant salamander cell line (GSM) was
AdBid sequence analysis
AdBid consists of 191 amino acid residues with a calculated molecular weight of 21.3 kDa and a theoretical isoelectric point (pI) of 4.93 (Figure 1A). Like other Bids, AdBid has a BH3 interaction domain (BID) (residues 2–185) (Figure 1B). Sequence alignments showed that AdBid shares 24%, 24%, 27%, 27%, 28%, 30%, 34%, 37%, 50% and 52% of total sequence identity with Bid homologues from fish and amphibian species includingRhincodon typus,Danish Rerio,Poeciliopsis fertile,
The Bcl-2 (B-cell lymphoma-2) superfamily is a class of proteins primarily involved in the intrinsic pathway of apoptosis, sharing conserved sequences (~20 residues) known as Bcl-2 (BH) domains or homology motifs (designated BH1, BH2, BH3 and BH4 corresponding to α-helical segments) (Sato et al., 1994; Adams and Cory, 1998). Bid, a BH3-interacting domain death agonist, is a proapoptotic member of the BH3-only Bcl-2 superfamily and as such has intracellular targeting capabilities
The work was supported by grants fromLife Sciences Foundation of Hubei Provincefrom the chin (2020CFB347) iCentral scientific institution of public interest Foundation for fundamental research,CAFS(2020XT0401).
Amphibian invitromes: past, present and future contributions to our understanding of amphibian resilience
2023, Developmental and comparative immunology
Around the world, many amphibian populations are in decline, and infectious diseases are the main cause. Given the enormous threat that infectious diseases pose to amphibian populations, there is a need to understand the host-pathogen-environment interactions that govern amphibian susceptibility to disease and mortality. However, the use of animals in research raises an ethical dilemma, which is complicated by althe rate at which many amphibian populations decline. Therefore,in vitroresearch systems such as cell lines are valuable tools for advancing our understanding of the amphibian immune system. In this review, we compile a list of established amphibian cell lines (amphibian invitromes) to date, highlighting how research using amphibian cell lines has increased our understanding of the amphibian immune system, ranavirus defense, andBatrachochytrium dendrobatidisreplication in host cells and represent our view of how the future use of amphibian cell lines could advance the field of amphibian immunology.
Establishment and characterization of a cell line from the brain of Japanese flounder (Paralichthys olivaceus) and its application in the study of viral infections
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It is the main pathogen affecting the Chinese giant salamander and leading to severe disease in this endangered species (Ma et al., 2014). Only a limited number of susceptible cell lines have been reported for both viruses (Ariel et al., 2009; Li et al., 2014; Li et al., 2021). Therefore, the JFB cell line we created here provides a new in vitro model for BIV and GSIV studies, especially for virus propagation and preparation of virus vaccines and study of virus-host cell interactions.
Fish cell lines are an important tool in many biological studies. Here, we generated a new Japanese flounder (JFB) cell line from the brain tissue of Japanese flounder (Paralichthys olivaceus), which was successfully cultured for more than 120 passages and consisted mainly of fibroblast-like cells. Sequencing of the actin gene confirmed that JFB cells were derived from Japanese flounder, and JFB was also identified as a neural stem cell line based on nestin gene mRNA expression. JFB cells could grow at temperatures from 17 to 29°C, with an optimum temperature of 23°C. Karyotype analysis showed that the number of chromosome modifications is 48, which indicates a normal diploid number of chromosomes. The transfection efficiency of pEGFP-N1 in JFB cells was up to 30%. JFB cells were susceptible to Hirame rhabdovirus (HIRRV), Lymphocystis virus virus (LCDV), Bohle virus (BIV) and Chinese giant salamander iridovirus (GSIV), as shown by varying degrees of cytopathic effects. Viral infection or poly(I:C) stimulation increased the expression of many genes associated with the immune system, includingIL-1β,IL8,MXandTNF. These results indicated that the newly established JFB cell line is an ideal tool for studying gene manipulation, host-virus interaction and potential vaccine development.
Zebrafish BID exerts an antibacterial role by negatively regulating p53, but in a caspase-8-independent manner
2021, Frontiers in immunology
Featured Articles (6)
Identification and analysis of two lebocins in the oriental armyworm Mythimna separata
Developmental and Comparative Immunology, Volume 116, 2021, Article 103962
The insect immune system can produce defense molecules such as antimicrobial peptides (AMPs) to eliminate invading pathogens. Here we report the identification of two cDNAs (MseLeb1,MseLeb2), which encodes the lepidopteran lebocin preprotein in the oriental worm,Mythymna separated. Their open reading frames are 483/492 bp and encode peptides of 161/164 amino acids.MseLeb1it is expressed mainly in adipose tissue and epidermis, whileMseLeb2it is mainly found in the fat body, Malpighian tube and epidermis. They were significantly encouragedEscherichia coli,Staphylococcus aureus, andBeauveria bassianain hemocytes.Preproteins can be processed after the RXXR motif into mature peptides. Multiple sequence alignments indicate that MseLeb1 (18-42, 121-161) are potentially active peptides. Five peptides were synthesized for analysis: 18-42, 121-161, 121-154, 121-151, 121-146. Synthetic peptides showed agglutination activity, but not hemolytic activity. Bacterial growth test, colony formation test and electron microscopy showed that the synthetic peptides can inhibit bacterial growth and destroy the bacterial cell wall.B. bassianaconidia and blastospores were lysed with synthetic peptides. These results indicate that MseLeb1 and MseLeb2 are immunologically responsive lebocins and that the mature peptides have antibacterial and antifungal activity.
MicroRNA repertoire and comparative analysis of ranavirus-infected Andrias davidianus using deep sequencing
Developmental and comparative immunology, volume 85, 2018, p. 108-114 (view, other).
Andrias davidianusis a large and economically important amphibian in China. Ranavirus infection causes serious losses indavidianusagricultural industry. MicroRNA-mediated host-pathogen interactions are important in antiviral defense. In this study, five small RNA libraries from infected and uninfected ranavirusdavidianusspleens were sequenced using high-throughput sequencing. The miRNA expression pattern, potential functions and target genes were investigated. A total of 1356 known and 431 new miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes were associated with apoptosis, signaling pathway, and immune response categories. The analysis identified 82 down-regulated and 9 up-regulated miRNAs with differential expression, whose putative target genes are involved in pattern recognition and immune response receptor signaling pathways. These findings suggest that miRNAs play a key role indavidianusresponse to ranavirus and can be used to further functionally identify miRNAs, leading to new approaches for improvementsdavidianusresistance to ranaviruses.
CqPP2A inhibits white macule syndrome virus infection by enhancing antimicrobial expression in red claw carcinomas of Cherax quadricarinatus
Developmental and Comparative Immunology, Volume 116, 2021, Article 103913
Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells that is responsible for most of the serine/threonine phosphatase activity in cells involved in the regulation of immune signaling pathways and antiviral responses. However, most research on PP2A has been conducted in mammals and little in crustaceans. In this study, two subunits of PP2A (named asCqPP2AbandCqPP2Ac) were characterized as being involved in white spot syndrome virus (WSSV) infection of hematopoietic tissue (Hpt) cells in red claw carcinomasCherax quadricarinatus. Open reading frame (ORF).CqPP2Abwas 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORFCqPP2Acwas 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript fromCqPP2AbandCqPP2Acboth were widely expressed in all tissues tested with highest expression in hemocytes followed by high expression in Hpt. Gene expressionCqPP2AbandCqPP2Acboth were significantly reduced 6 h after infection with WSSV (6 hpi) in Hpt cells. It is important that viral immediate early gene expressionIE1and the late envelope protein of the viral geneVP28both were significantly increased after WSSV infection after gene silencingCqPP2AbLubCqPP2Acin Hpt cells, suggesting thatCqPP2AbandCqPP2Accan inhibit WSSV infection in Hpt cells, possibly by increasing the expression of antimicrobial substances, given the significantly reduced expression of antilipopolysaccharide factor, shell and lysozyme after gene silencingCqPP2AbLubCqPP2Acappropriate. These findings shed new light on the mechanism of WSSV infection and the antiviral response in crustaceans.
Purification of secreted lectin from the skin of Andrias davidianus and its antibacterial activity
Comparative Biochemistry and Physiology, Part C: Toxicology and Pharmacology, Volume 167, 2015, p. 140-146
Lectin secreted fromAndrias davidianusskin (ADL) was purified by affinity chromatography on porcine gastric mucin (type III) cross-linked albumin (PSM), followed by gel filtration on Sephadex G-100 and HPLC on a TSK G3000PW gelXL. The purified lectin was found to be a dimeric protein as shown by SDS-PAGE and MALDI-TOF analyses. SDS-PAGE showed that the ADL protein has a molecular weight of 17 kDa. ADL produced a band of 8.5 kDa when examined by SDS-PAGE under reducing conditions. Agglutinated native and trypsinized human ADL erythrocytes. Hemagglutination activity was inhibited by glycoproteins such as PSM and asialo-PSM, but not by any of the tested monosaccharides. The activity was stable from 4°C to 50°C. Significant ADL activity was observed in the pH range of 4-5. The lectin reaction did not depend on the presence of divalent Ca cation2+Mg2+. The N-terminal sequence of ADL was found to be VGYTVGATPM. The lectin showed antibacterial activity by inhibiting growth and respirationEscherichia coli,Enterobacter aerogenes,Staphylococcus aureus,Bacillus subtilisandShewanellasp. In addition, ADL had an inhibitory effect on yeastSaccharomyces cerevisiae. These findings suggest that ADL plays an important role in innate immunityA. davidianuson the surface of the body.
Extensive MHC diversity in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel assembly variants
Developmental and Comparative Immunology, Volume 42, Issue 2, 2014, p. 311-322 (view, other).
A number of MHC alleles (including 26 class IA, 27 class IIA and 17 class IIB) have been identified from the Chinese giant salamanderAndrias davidianus(Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, derived tertiary structures and genetic diversity. Most of the variation between alleles occurs in the putative peptide binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC alleles IA, IIA and IIB is shown: one full-length transcript and one new spliced variant. Despite the pool of external domains, these variants show similar subcellular localization to the full-length transcripts. In addition, the expression of MHC isoforms is up-regulatedLiveandin vitrostimulation withAndrias davidianusranavirus (ADRV), indicating their potential role in the immune response. The results provide insight into the variability and MHC function of this ancient and endangered urodele amphibian.
Involvement of short-type peptidoglycan recognition protein (PGRP) from the Chinese giant salamander Andrias davidianus in the immune response to bacterial infection
Developmental and comparative immunology, volume 88, 2018, p. 37-44 (view, other).
PGRPs (peptidoglycan recognition proteins) can recognize peptidoglycan and play an important role in innate immunity in various animals. To date, the functions of PGRP have been studied in various animals, but few reports have been made on amphibian PGRP. The current study identified a short form of PGRP from the Chinese giant salamander and investigated its contribution to innate immunity. ORF zNoticeThe PGRP-SC2 cDNA was 573 bp, encoded 190 amino acids and contained the PGRP domain and amidase_2. qPCR analysis showed thisNoticePGRP-SC2 mRNA transcripts are expressed in various tissues, with the highest expression levels in muscle, intestine, and spleen. The results of peptidoglycan (PGN) immunochallenge showed that the expression patternsNoticePGRP-SC2 was significantly elevated in erythrocytes and spleen early in the injection. Recombinant protein AdPGRP-SC2, which can have binding affinity to different bacteria, was produced and purified. In the presence of Zn2+, rAdPGRP-SC2 can exhibit broad PAMP binding activity, strongly agglutinate bacteria, and exhibit amidase enzymatic activity. Collectively, these data indicate that AdPGRP-SC2 can act as a PRR to recognize invading microorganisms and as an antimicrobial effector during the innate immune response.A. davidianus.
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