Zhiming Xiang

Characteristic and Functional Analysis of Toll-like Receptors (TLRs) in the lophotrocozoan, Crassostrea gigas, Reveals Ancient Origin of TLR-Mediated Innate Immunity

The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostrea gigas (CgTLRs). All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C . gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.

Proteomic basis of stress responses in the gills of the pacific oyster crassostrea gigas

The Pacific oyster Crassostrea gigas is one of the dominant sessile inhabitants of the estuarine intertidal zone, which is a physically harsh environment due to the presence of a number of stressors. Oysters have adapted to highly dynamic and stressful environments, but the molecular mechanisms underlying such stress adaptation are largely unknown. In the present study, we examined the proteomic responses in the gills of C. gigas exposed to three stressors (high temperature, low salinity, and aerial exposure) they often encounter in the field. We quantitatively compared the gill proteome profiles using iTRAQ-coupled 2-D LC-MS/MS. There were 3165 identified proteins among which 2379 proteins could be quantified. Heat shock, hyposalinity, and aerial exposure resulted in 50, 15, and 33 differentially expressed gill proteins, respectively. Venn diagram analysis revealed substantial different responses to the three stressors. Only xanthine dehydrogenase/oxidase showed a similar expression pattern across the three stress treatments, suggesting that reduction of ROS accumulation may be a conserved response to these stressors. Heat shock caused significant overexpression of molecular chaperones and production of S-adenosyl-l-methionine, indicating their crucial protective roles against protein denature. In addition, heat shock also activated immune responses, Ca(2+) binding protein expression. By contrast, hyposalinity and aerial exposure resulted in the up-regulation of 3-demethylubiquinone-9 3-methyltransferase, indicating that increase in ubiquinone synthesis may contribute to withstanding both the osmotic and desiccation stress. Strikingly, the majority of desiccation-responsive proteins, including those involved in metabolism, ion transportation, immune responses, DNA duplication, and protein synthesis, were down-regulated, indicating conservation of energy as an important strategy to cope with desiccation stress. There was a high consistency between the expression levels determined by iTRAQ and Western blotting, highlighting the high reproducibility of our proteomic approach and its great value in revealing molecular mechanisms of stress responses.

Genomic characterization and expression analysis of five novel IL-17 genes in the Pacific oyster, Crassostrea gigas

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in clearing extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. In the present study, five novel IL-17 homologs were identified by searching and analyzing the Pacific oyster genome. All six CgIL-17 members (including a previously reported homolog) contained four conserved cysteines that were used in the formation of disulfide bonds. Phylogenetic analysis showed that all invertebrate IL-17s were clustered into one group, implying that invertebrate IL-17s evolved from one common ancestral gene and subsequently diversified. All CgIL-17s shared the same genomic structure, containing two exons and one intron, except for the CgIL-17-3 and CgIL-17-5 genes, which each had only one exon. The expression pattern of the CgIL-17 genes was analyzed by qRT-PCR in a variety of tissues and at different developmental stages, and these genes were highly expressed in the gill and digestive gland tissues. Moreover, the expression of the CgIL-17 family genes was significantly up-regulated in hemocytes challenged with Pathogen-Associated Molecular Patterns (PAMPs). CgIL-17-3 had a strong response to lipopolysaccharide (LPS) and heat-killed Vibrio alginolyticus (HKVA) challenge, while CgIL-17-5 and CgIL-17-6 can be activated by peptidoglycan (PGN), but not by heat-killed Listeria monocytogenes (HKLM). The distinct, up-regulated transcript levels of the CgIL-17s in response to PAMPs challenge further indicate that CgIL-17s are likely to be significant components of immune responses by playing diversified roles in host defense in the Pacific oyster. These findings suggest that CgIL-17s are involved in innate immune responses and further supports their conserved function in mollusks immunity.

Administration of recombinant IFN1 protects zebrafish (Danio rerio) from ISKNV infection.

In the present study, we used the infectious spleen and kidney necrosis virus (ISKNV) and zebrafish model system to investigate the inhibitory effect of recombinant zebrafish interferon 1 (zfrIFN1) on acute viral infection and the impact of time of zfrIFN1 administration on its protective efficacy. In vivo experiments showed that administration of recombinant zfrINF1 up-regulated expression of several IFN-stimulated genes within 24 h of injection, and expression levels of these genes dropped to normal levels similar to those in control fish within three days. However, the transcriptions of two viral genes, the major capsid protein and virus protein 48 genes, were significantly inhibited for at least three days, indicating a longer duration of the zfrIFN1-mediated innate immune effect. To evaluate the protective efficacy of zfrIFN1 against ISKNV infection, we compared the relative percentage survival (RPS) of ISKNV-infected zebrafish by intraperitoneally (IP) injecting the fish with zfrIFN1 at different time points before or after infection. IP injection with 1 microg zfrIFN1/g fish body weight at 24, 6 or 0 h before virus infection or 6 h after virus infection significantly improved fish survival. However, IP injection with an equal dose of zfrIFN1 24 h post-infection did not provide significant protection to the fish. Our results suggest that zfrIFN1 is potent in inhibiting ISKNV acute infection and initiating the innate immune response in zebrafish, but its efficiency depends on the time of administration. This study shows the protective effects of interferon against a DNA-virus in fish for the first time and provides information about the efficacy of fish interferon that will prove useful in possible therapeutic applications.

Characteristics of the interferon regulatory factor pairs zfIRF5/7 and their stimulation expression by ISKNV Infection in zebrafish (Danio rerio).

The interferon regulatory factor (IRF) family plays critical roles in a hosts virus infection responses. In this study, two IRF family members, zfIRF5 and zfIRF7, are identified in zebrafish. The zfIRF5 protein encodes 297 amino acids without the carboxyl IRF3 domain. We suggest that zfIRF5 is a new kind of splicing variant, following the nine other kinds of IRF5 splicing variants found in mammals. The zfIRF7 protein is identified as a member of the IRF7 family, compared to the human IRF7 protein, the amino acid sequence of zfIRF7 only with 29% identity and devoid a virus activated domain (VAD). There zfIRF5/7 proteins are expressed in all 11 selective zebrafish tissues within 6-120h of embryonic development. Laser confocal microscopy shows that the full length the proteins are separately located in the cytoplasm. Mutation experiments show that the nuclear localization signals (NLS) of zfIRF7 and zfIRF-5 are at the N-terminal and C-terminals, respectively. In the assays, zfIRF7 expression increases during infectious spleen and kidney necrosis virus (ISKNV) infection and by poly(I:C) and LPS injections, both of which activate the transcriptional activity of L8G5-luc plasmids. The over-expression of zfIRF5/7 activates the interferon-stimulated response elements (ISRE) signal pathway. In addition, zfIRF7 can activate IFN-β, zfIRF5/7. Co-immunoprecipitation assays and laser co-confocal microscopy show that the two proteins could interact, and zfIRF7 may stimulate zfIRF5 to move into the nucleus. The co-expression of zfIRF5/IRF7 suppresses the transcriptional activities of IFN-β in HEK293T cells.

Characterization of a TnMAVS protein from Tetraodon nigroviridis

A growing family of cellular proteins encoding for caspase activation and the recruitment domain (CARD) plays a crucial role in immunity by sensing viral infections and signaling antiviral immune defenses. We obtained a MAVS-like protein (named TnMAVS) from Tetradon nigroviridis, which contains a CARD domain, a pro-rich domain, and a TM domain similar to human MAVS. A fluorescence assay showed that TnMAVS was located in the cytoplasm and near by the membrane, and not the mitochondria in FHM cells. As such, it was considered as a new member of MAVS. The TnMAVS was highly expressed in the liver and muscle of T. nigroviridis. In the spleen, TnMAVS was down-regulated when the fish was treated with polyinosinic:polycytidylic acid or challenged with ISKNV, but was not affected by PGN or LPS. The dual luciferase reporter assay revealed that TnMAVS overexpression resulted in the activation of the interferon-sensitive response element and NF-κB signal pathways. In addition, a characteristic TRAF3-associated peptide PVQD was found in the TnMAVS sequence. Co-immunoprecipitation assays indicated that TnMAVS could interact with zfTRAF3 in eukaryotic cells.

Activator protein-1 (AP-1) and response to pathogen infection in the Hong Kong oyster (Crassostrea hongkongensis).

Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells.

Cloning, characterization and expression analysis of a caspase-8 like gene from the Hong Kong oyster, Crassostrea hongkongensis

Apoptosis plays a key role in many biological processes, including homeostasis within the immune system. A family of cysteine proteases, the caspases, constitutes the core of the apoptotic machinery. We have characterized the first bivalve caspase-8 ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChCaspase-8). The full-length cDNA is 1945 bp in length encoding a putative protein of 557 amino acids that contains two N-terminal DED domains, and a CASc domain at the C-terminus. ChCaspase-8 is ubiquitously expressed in oysters, with highest expression levels in the gonad and labial palps. Following microbial infection, the expression of ChCaspase-8 increased in hemocytes from 12 to 72 h post-challenge. When expressed in HeLa cells, ChCaspase-8 is located in the cytoplasm, while over-expression of ChCaspase-8 in HEK293T cells activates the transcriptional activities of NF-κB. These results indicate that ChCaspase-8 might play an important role in the immune and apoptotic responses of oysters.

Expression and function analysis of two naturally truncated MyD88 variants in the Pacific oyster Crassostrea gigas

Myeloid differentiation factor 88 (MyD88) is the classic signaling adaptor that mediates Toll/interleukin-1 receptor (TIR/IL-1R) dependent activation of nuclear factor-kappa B (NF-κB). In this study, two naturally truncated MyD88 members were identified from the Pacific oyster (Crassostrea gigas), namely CgMyD88-T1 and CgMyD88-T2. The full-length cDNA of CgMyD88-T1, CgMyD88-T2 are 976 bp and 1038 bp in length, containing an ORF of 552 bp and 555 bp, respectively. The two ORF encode a putative protein of 183 and 184 amino acids, respectively, with a calculated molecular weight of about 21 and 22 kDa. When compared to complete MyD88 paralogues, we found that both CgMyD88-T1 and CgMyD88-T2 contain only TIR domain but lack DD (Death Domain), which share 90.8% of similarity and 71.7% of identity with each other. Phylogenetic tree demonstrated that CgMyD88-T1 and CgMyD88-T2 clustered together and belonged to mollusk branch. Meanwhile, genomic arrangement analysis displayed that the two truncated MyD88s were distributed in tandem in one scaffold, revealing that they may originate from one truncated MyD88 ancestor recently. Expression profile showed that both of CgMyD88 variants were ubiquitously expressed in all tested tissues with highest expression in the gills and hemocytes, respectively. Both truncated CgMyD88 mRNAs were significantly up-regulated in hemocytes under HKLM (heat-killed Listeria monocytogenes) and HKVA (heat-killed Vibrio alginolyticus) challenge. Moreover, either CgMyD88-T1 or CgMyD88-T2 were able to inhibit MyD88 activated Rel/NF-κB activity in HEK293 cell, demonstrating their negative role in regulating MyD88-mediated immune signaling.

A novel molluscan Fos gene with immune defense function identified in the Hong Kong oyster, Crassostrea hongkongensis.

The transcription factor Fos is a member of one of the best-studied AP-1 sub-families and has been implicated in a wide variety of biological processes, including the regulation of apoptosis, immune responses and cytokine production. In this report, a novel mollusk Fos (referred to as ChFos) gene was cloned and characterized from the Hong Kong oyster, Crassostrea hongkongensis. The deduced ChFos protein sequence comprised 333 amino acids and shared significant homology with invertebrate homologs. Phylogenetic analysis revealed that ChFos clusters with Fos from Crassostrea gigas and Crassostrea ariakensis. Quantitative real-time PCR analysis revealed that ChFos mRNA was broadly expressed in all tested tissues and during different stages of the oysters embryonic and larval development. In addition, the expression of ChFos mRNA was significantly up-regulated under challenge with microorganisms (Vibrio alginolyticus, Staphylococcus haemolyticus and Saccharomyces cerevisiae) and pathogen-associated molecular patterns (PAMPs: LPS, PGN and polyI:C). Moreover, fluorescence microscopy showed that ChFos protein is localized in the nucleus in HEK293T cells. Reporter assays suggested that ChFos may act as an efficient transcription activator in the regulation of AP-1-responsive gene expression through interaction with ChJun. Overall, this study presents the first experimental evidence of the presence and functional characteristics of Fos in mollusks, which reveals its involvement in host protection against immune challenge in the oyster.