Fan Mao

A novel p38 MAPK indentified from Crassostrea hongkongensis and its involvement in host response to immune challenges

p38 mitogen-activated protein kinases (MAPKs) are conserved serine/threonine-specific kinases that are activated by various extracellular stimuli and play crucial regulatory roles in immunity, development and homeostasis. However, the function of p38s in mollusks, the second most diverse group of animals, is still poorly understood. In this study, a novel molluscan p38 (designated Chp38) was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA encoded a putative protein of 353 amino acids with a calculated molecular weight of approximately 40.3kDa. Similar to other reported p38 family proteins, the deduced Chp38 sequence contained a conserved dual phosphorylation TGY motif and a substrate binding site of ATRW. Phylogenetic analysis revealed that Chp38 was closest to its homolog from the Pacific oyster and belonged to the mollusk cluster. Quantitative real-time PCR analysis showed that Chp38 was constitutively expressed in all examined oyster tissues and developmental stages and that its expression in hemocytes was significantly up-regulated after pathogen (Vibrio alginolyticus and Staphylococcus haemolyticus) and PAMP (lipopolysaccharide and peptidoglycan) infections. Moreover, overexpression analysis revealed that Chp38 was localized in both the cytoplasm and nucleus of HEK293T cells and that it could significantly enhance AP-1 reporter gene activation in a dose-dependent manner. Altogether, these results provide the first experimental evidence of a functional p38 in oysters and suggest its involvement in the innate immunity of C. hongkongensis.

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in Crossastrea gigas

Abstract Tumor necrosis factor receptor‐associated factor 6 (TRAF6) has been demonstrated to be a key signaling molecule involved in adaptive and innate immunity. In this study, we obtained the full length CgTRAF6 cDNA and analyzed the characteristics of the ORF and the peptide sequence in Crassostrea gigas. The deduced protein sequence of CgTRAF6 includes a conserved C‐terminal TRAF domain following the RING and the zinc finger domain. The TRAF domain is composed of coiled‐coil TRAF‐N and MATH (meprin and TRAF‐C homology) subdomains. Furthermore, phylogenetic analysis revealed that CgTRAF6 is clustered together with other members TRAF6 family and is placed in a sub‐cluster singly which had a close relationship with Drosophila melanogaster. Expression analysis of CgTRAF6 indicated its constitutive expression in all tissues including mantle, adductor muscle, digestive tract, gonads, heart, gill, and hemocyte. Immune challenge with Vibrio alginolyticus and poly I:C resulted in significant up‐regulation of CgTRAF6 expression. Dual‐luciferase reporter assays showed that CgTRAF6 could activate both pNF‐&kgr;B‐Luc and pISRE‐Luc expression, suggesting CgTRAF6 is potentially involved in NF‐&kgr;B and the interferon signaling pathway. Furthermore, RNAi mediated knockdown of CgTRAF6 resulted in the down‐regulation of several putative anti‐viral signaling (IRF) and effector (PKR & Viperin) molecules coding genes, 7 days post‐injection. These results collectively indicate that CgTRAF6 is a member of TRAF6 sub‐family and is potentially involved in immune defense system against invading bacteria and viruses in Crassostrea gigas. HighlightsA gene from Crassostrea gigas encoding TRAF6 was identified.Expression of CgTRAF6 was identified in all tissue and changed after immune challenge.CgTRAF6 can effectively activate NF‐&kgr;B reporter and act as a key signaling molecule in NF‐&kgr;B pathway.

A molluscan extracellular signal-regulated kinase is involved in host response to immune challenges in vivo and in vitro

ABSTRACT Extracellular signal‐regulated kinases (ERKs) are a group of highly conserved serine/threonine‐specific protein kinases that function as important signaling intermediates in mitogen‐activated protein kinase (MAPK) pathways, which are involved in a wide variety of cellular activities, including proliferation, inflammation and cytokine production. However, little is known about the roles of this kinase in mollusk immunity. In this study, we identified a molluscan ERK homolog (ChERK) in the Hong Kong oyster (Crassostrea hongkongensis) and investigated its biological functions. The open reading frame (ORF) of ChERK encoded a polypeptide of 365 amino acids, with a predicted molecular weight of 41.96 kDa and pI of 6.43. The predicted ChERK protein contained typical characteristic motifs of the ERK family, including a dual threonine‐glutamate‐tyrosine (TEY) phosphorylation motif and an ATRW substrate binding site. Phylogenetic analysis revealed that ChERK belonged to the mollusk cluster and shared a close evolutionary relationship with ERK from Crassostrea gigas. In addition, quantitative real‐time PCR analysis revealed that ChERK expression was detected in all of the examined tissues and stages of embryonic development; its transcript level was significantly induced upon challenge with bacterial pathogens (Vibrio alginolyticus and Staphylococcus haemolyticus) in vivo and PAMPs (lipopolysaccharide and peptidoglycan) in vitro. Moreover, ChERK was mainly located in the cytoplasm of HEK293T cells. Taken together, these findings may provide novel insights into the functions of molluscan ERKs, especially their roles in response to immune challenge in oyster. HighlightsA novel oyster ERK was first identified from Crassostrea hongkongensis.ChERK shared a significant homology with other mollusk ERK proteins.ChERK was constitutively expressed in all examined oyster tissues and developmental stages.ChERK expression was significantly induced in response to bacterial pathogens and PAMPs challenges.ChERK was found to be located mainly in the cytoplasm of HEK293T cells.

The first invertebrate NFIL3 transcription factor with role in immune defense identified from the Hong Kong oyster, Crassostrea hongkongensis

Abstract NFIL3 (nuclear factor interleukin 3‐regulated) is a basic leucine zipper type transcription factor that mediates a variety of immune responses in vertebrates. However, the sequence information and function of NFIL3 homologs in invertebrates, especially mollusks, remains unknown. In the present study, the first NFIL3 homolog was identified in a marine mollusk, Crassostrea hongkongensis (designated as ChNFIL3), followed by its functional characterization. The full‐length cDNA of ChNFIL3 is 2221 bp and consists of an open reading frame (ORF) of 1536 bp that encodes a polypeptide of 551 amino acids. Simple Modular Architecture Research Tool (SMART) analysis indicated that ChNFIL3 has two basic leucin zipper domains, similar to the other known NFIL3 family proteins. Tissue distribution analysis of NFIL3 in this mollusk revealed high expression in digestive glands and hemocytes. A significant induction in the mRNA level of ChNFIL3 was observed following bacterial stimulation. ChNFIL3 was found to be localized in the nucleus and over expression of ChNIFL3 led to upregulation of transcriptional activity of an NF‐&kgr;B reporter gene in HEK 293T cells, indicating its role in innate immunity. Furthermore, addition of exogenous recombinant ChNFIL3 proteins resulted in enhanced mRNA level of hemocyte interleukin 17 in vitro. In conclusion, our findings revealed that NFIL3 in molluscs, plays a conserved role in host defense, similar to its mammalian homolog. HighlightsChNFIL3 was involved in the bacterial infection of oysters.ChNFIL3 could enhance the expression of interleukin 17 in vitro.Over expression of ChNFIL3 resulted in up‐regulated the transcriptional activity of an NF‐&kgr;B reporter gene.

A Lysin motif (LysM)-containing protein from Hong Kong oyster, Crassostrea hongkongensis functions as a pattern recognition protein and an antibacterial agent

Lysin motif (LysM)-containing proteins are a family of carbohydrate-binding modules and are generally regarded as chitin- and peptidoglycan-binding proteins. In the present study, a novel LysM-containing protein, designated as ChLysM, was cloned and identified in a marine mollusk, Crassostrea hongkongensis. The full-length cDNA of ChLysM consists of 1129 bp, with an open reading frame of 861 bp encoding a 286 amino acid polypeptide. The deduced protein had a calculated molecular mass of 32.66 kDa and a pI of 8.16. SMART analysis indicated that ChLysM has one Lysin motif and a transmembrane region in the C-terminal residues. Tissue distribution analysis of ChLysM revealed high expression in gills and hemocytes. The upregulated transcripts of ChLysM in response to bacterial challenge suggest that ChLysM is involved in innate immunity against pathogen infection. The recombinant protein of ChLysM was found to bind to various kinds of peptidoglycans from Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae, as well as binding strongly to both Gram-positive and Gram-negative bacteria. Moreover, ChLysM displayed broad-spectrum antibacterial activity against both Gram-positive bacteria (S. aureus and S. haemolyticus) and Gram-negative bacteria (Escherichia coli and Vibrio alginolyticus). Collectively, these results indicate that ChLysM is a pattern recognition molecule with bacterial growth-inhibiting activity in immune defense of C. hongkongensis.

The Molecular Mechanism Underlying Pro-apoptotic Role of Hemocytes Specific Transcriptional Factor Lhx9 in Crassostrea hongkongensis

Hemocytes are the central organ of immune defense against pathogens by means of inflammation, phagocytosis, and encapsulation in mollusks. The well-functioning of the host immune system relies on the hemocytes’ task exertion and frequent renewal, but the underlying renewal mechanism remains elusive at the gene level. Here, we identified one transcription factor, LIM homeobox 9, in Crassostrea hongkongensis (ChLhx9) that could be involved in hemocyte apoptosis or renewal. ChLhx9 contains a homeodomain and two LIM domains. The expression profile of ChLhx9 showed that it was specific and had high expression in hemocytes, and it significantly increased under the bacterial challenge. RNA interference of ChLhx9 dramatically decreased the apoptosis rate of hemocytes when compared with a control group, which strongly implies its pro-apoptotic role in hemocytes. Furthermore, the genomic responses to the knockdown of ChLhx9 were examined through RNA-seq, which showed that multiple pathways associated with cell apoptosis, including the apoptosis pathway, hippo signal pathway and p53 signaling pathway, were significantly down-regulated. Meanwhile, seven of the key apoptotic genes were confirmed to be upregulated by ChLhx9, among which ChASPP1 (apoptosis stimulating protein of p53) was confirmed to induce hemocyte apoptosis strongly, which demonstrates that ChASPP1 was a downstream target mediated by ChLhx9 that caused apoptosis. In conclusion, tissue-specific transcription factor ChLhx9 induces hemocyte apoptosis through activating apoptotic genes or pathways, which could contribute to hemocyte renewal and immune defense in oysters.

Dual roles of cystatin A in the immune defense of the pacific oyster, Crassostrea gigas

ABSTRACT Cystatins are a large family of the proteins that function as reversible and tight‐binding inhibitors of cysteine proteases, which consequently regulate multiple physiological activities including apoptosis and innate immunity. In the present study, we cloned a gene from Crassostrea gigas encoding cystatin, which is related to cystatin A superfamily. CgCytA was comprised of a cystatin‐like domain with two conserved glycine residues (GG) near the N‐terminal and a highly conserved glutamine‐valine‐glycine (Q‐X‐V‐X‐G) motif in the form of QVVAG loop. Transcription analysis of CgCytA indicated its constitutive expression in all tissues including mantle, gill, digestive tract, hemocytes, heart, adductor muscle, and gonads. Immune challenge with Vibrio alginolyticus, resulted in significant down‐regulation of CgCytA expression at the initial stages of infection (till 12 h post infection) and the expression of cystatin increased 48 h post infection. Protease assay demonstrated the concentration of cystatin needed to inhibit half of the maximum biological response of cysteine protease is 14.4 &mgr;g/L (IC50). Furthermore, RNAi of CgCytA resulted in increase of apoptotic cell population in hemocytes of C. gigas, suggesting protection role of CgCytA from hemocytes apoptosis. Unexpectedly, knockdown of CgCytA leaded to enhancement of bacterial clearance in vivo, implying that CgCytA may negatively regulate immune defense by suppressing endogenous cysteine protease. Therefore, CgCytA plays dual roles in protection of host hemocytes from apoptosis and control of bacterial clearance, which may server as one of key endogenous balancer between apoptosis and innate immunity in oyster. HighlightsOne homolog of cystatin was identified from Crassostrea gigas.CgCytA mRNA fluctuated after bacterial challenge.Recombinant CgCytA could effectively inhibit the activity of cysteine proteases.Knockdown of CgCytA leaded to increase of hemoctyes apoptosis and bacterial clearance in vivo.

The first morphologic and functional characterization of hemocytes in Hong Kong oyster, Crassostrea hongkongensis

&NA; Hemocytes are the first line of defence of the innate immune system of molluscs. For the first time hemocytes of Crassostrea hongkongensis were morphologically and functionally characterized, identifying circulating cell types and studying their involvement in immune responses. In the present study, two main populations, hyalinocytes and granulocytes, were characterized based on the presence or absence of cytoplasmic granules, using light and electron microscopy (TEM), and flow cytometry analyses. Granulocytes are 7–13 &mgr;m in diameter and present evident cytoplasmic granules, and hyalinocytes, 6–15 &mgr;m in diameter, with a few or no granules. The mean number of circulating hemocytes in the hemolymph was 2.52 × 106 cells/mL. Flow cytometry indicated that both granulocytes and hyalinocytes showed cell phagocytosis and reactive oxygen species (ROS) production. However, phagocytosis and spontaneous production of reactive oxygen species (ROS) in granulocytes are much more active compared with hyalinocytes, which demonstrated that the granulocytes are the main hemocytes involved in the immune response of Hong Kong oyster. Moreover, the cell‐free hemolymph showed antibacterial activity against Vibrio alginolyticus. Our results provide the basic information of hemocytes population of Hong Kong oyster for further investigations associated with innate immunity. HighlightsHemocytes from Crassostrea hongkongensis were separated into two groups, hyalinocytes and granulocytes.Granulocytes are much more active in the cell phagocytosis and ROS production compared with hyalinocytes.Hemolymph showed antibacterial activity with Vibrio alginolyticus.

Structural and functional analysis of interferon regulatory factors (IRFs) reveals a novel regulatory model in an invertebrate, Crassostrea gigas

Abstract Interferon regulatory factors (IRF), a family of transcription factors, are involved in the regulation of interferon to response the pathogen infection. Here, three IRF‐like genes including CgIRF1a, CgIRF1b and CgIRF8 were identified in the genome of the oyster C. gigas. Among these genes, CgIRF1a and CgIRF1b, which are tandemly located in adjacent loci of scaffold 4, share the same domains. Phylogenetic analysis indicated that CgIRF1a and CgIRF1b were two paralogs that may originate from duplication of the same ancestral IRF gene. Subcellular localization analysis confirmed the nuclear distribution of CgIRF1a and CgIRF1b. Dual‐luciferase reporter assay showed that CgIRF1a significantly activated the ISRE reporter gene, whereas CgIRF1b did not. Additionally, overexpression of CgIRF1b could significantly suppress the activation effect of CgIRF1a, which strongly suggests that CgIRF1b may serve as a regulator of the IRF signaling pathway. Furthermore, the result of native page revealed that CgIRF1a would form homologous dimers, and CgIRF1b would interact with CgIRF1a to inhibit the activity of the latter. Taken together, one novel regulatory model of IRF signaling pathways has been raised one paralog of IRF has evolved and appears to be a regulator of IRF. HighlightsThree genes from Crassostrea gigas encoding IRFs were identified.CgIRF1a and CgIRF1b were two paralogs which were tandemly located in adjacent loci and shared the same domains.CgIRF1a significantly activated the ISRE reporter gene.CgIRF1b may serve as one regulator of the IRF signaling pathway by effectively inhibiting the activity of CgIRF1a.

Characterization and Identification of Differentially Expressed Genes Involved in Thermal Adaptation of the Hong Kong Oyster Crassostrea hongkongensis by Digital Gene Expression Profiling

Thermal exposure of sessile marine animals inhabiting estuarine intertidal regions is a serious concern with respect to their physiological processes. Organisms living in this region can be exposed to high temperature (>40 °C) during the low tide, which may affect the survival of these organisms. The Hong Kong oyster, Crassostrea hongkongensis, distributed along the coast waters of the South China Sea, is one of the dominant sessile inhabitants of marine intertidal region which undergoes large seasonal temperature fluctuations (up to 10-20 °C during diurnal cycles) every year. To cope with acute thermal stress, it has developed several adaptation mechanisms, eg the firmly shut of the shells. However, the genetic basis of these mechanisms remain largely unclear. To better understand how acute thermal exposure affects the biology of the oyster, two cDNA libraries obtained from the gill of oysters exposed to 37 °C thermal stress and ambient temperature were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. In total, 5.9 and 6.2 million reads were obtained from thermal stress and control libraries respectively, with approximately 74.25% and 75.02% of the reads mapping to the C. hongkongensis reference sequence. A total of 605 differentially expressed transcripts could be detected in the thermal stress group as compared to the control group, of which 378 are up-regulated and 227 are down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these Differentially Expressed Genes (DEGs) were enriched with a broad spectrum of biological processes and pathways, including those associated with chaperones, antioxidants, immunity, apoptosis and cytoskeletal reorganization. Among these significantly enriched pathways, protein processing in the endoplasmic reticulum was the most affected metabolic pathway, which plays an important role in the unfolded protein response (UPR) and ER-associated degradation (ERAD) processes. Our results demonstrate the complex multi-modal cellular response to thermal stress in C. hongkongensis.