Genome-wide identification and characteristic analysis of PGRP gene family in Tegillarca granosa reveals distinct immune response of the invasive pathogen.

The peptidoglycan recognition proteins (PGRPs) are conserved innate immune molecular in invertebrates and vertebrates, which play important roles in immune system by recognize the peptidoglycans of bacterial cell walls. Although PGRPs have been extensively characterized in insects, a systematic analysis of PGRPs in bivalves is lacking. In the present study, the phylogenic relationships, gene structures and expression profiles of PGRPs in marine bivalves were analyzed. The results indicated that the most PGRPs of bivalves were predicted to degrade the peptidoglycans and prevent excessive immunostimulation of bacteria. In addition, the results of the present study showed that the protein diversity of PGRPs in most marine bivalves was mainly generated by the alternative splicing of genes, however the alternative splicing of PGRP gene family was absent in Tegillarca granosa. The differences of PGRPs might be related to the genetic and environmental differences of marine bivalves. Spatiotemporal expression profiling in T. granosa suggested that PGRPs play important roles in the immune response of invasive pathogens. The present study describes a comprehensive view of PGRPs in the blood clam T. granosa and provides a foundation for functional characterization of this gene family in innate immune of marine bivalves.

Effects of spawning stress on the immune capacity of blood cockle Tegillarca granosa occurring on the south coast of Korea.

Spawning in marine bivalves is a great energy-demanding process, and it often results in lethal and sublethal stresses during the post-spawning period, including depressed immune capacity. The blood cockle Tegillarca granosa (Linnaeus, 1758) distributes widely in silty-mud tidal flats on the south coast of Korea, and they spawn in late summer. To understand the impacts of spawning on immune parameters, we analyzed the total hemocyte count (THC), hemocyte mortality, phagocytosis capacity, and reactive oxygen species (ROS) production of T. granosa in pre-, and post-spawning condition using a flow cytometer. Histology indicated that the blood cockles occurring on the south coast of Korea ripe and ready to spawn in July, and they spawned in August and September. The THC in the blood cockle hemolymph declined from pre-spawning (1.2 × 108 cell mL-1) to post-spawning (0.9 × 108 cell mL-1), possibly due to the spawning stress and the massive infiltration of hemocytes in the gonad to phagocytose and resorb the residual gametes during the post-spawning period. The hemocyte mortality increased linearly from August (4.1%) to November (9.1%), as the histology revealed that the blood cockle completed spawning, and they resorbed the relict gametes. The granulocyte phagocytosis capacity declined dramatically from July (12.7%) to September (6.0%), when the cockles were engaged in active spawning. The flow cytometry revealed that the production of reactive oxygen species (ROS) from the granulocytes and the erythrocytes type II increased linearly from August (0.8-0.9 × 105 A U.) to December (2.1-2.8 × 105 A U.), which may cause stresses at a cellular level during this period. As the data indicated, spawning is a stressful activity inducing depressed immunological capacities in the blood cockles.

Functional characterization of a putative tumor necrosis factor superfamily member 10 in blood clam (Tegillarca granosa).

Tumor necrosis factor superfamily member 10 (TNFSF10), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Apo-2L, is one of the important members of the TNF superfamily. It is well demonstrated that TNFSF10 preferentially induces a variety of tumor cell apoptosis, and therefore exerts an important role in tumor immune surveillance. However, the function of TNFSF10 in pathogen defense is poorly understood, especially in invertebrates. The blood clam (Tegillarca granosa), an important commercial marine bivalve, plays an important ecological role in the marine ecosystem. The identification of immune genes will provide new perspective for disease control in the blood clam (T. granosa) farming. To better understand the biological function of TNFSF10 protein, the full-length cDNA of TNFSF10 homologous gene of T. granosa (TgTNFSF10) was cloned and identified for the first time, which was found to contain 1239 base pairs and encode 254 amino acids with a molecular weight of 29.5 kDa and a conserved TNF domain in the C-terminal. Quantitative RT-PCR analysis showed that TgTNFSF10 gene was constitutively expressed in all tested tissues, with the highest expression in hemocytes. LPS, Vibrio alginolyticus and Vibrio parahaemolyticus stimulations dramatically increased the expression of TgTNFSF10 in T. granosa (11.47-fold, 3.71-fold and 8.29-fold compared with the control respectively). In vitro experiments showed that recombinant TgTNFSF10 protein strongly inhibited the proliferation of HepG2 cells. Further confocal microscopy and flow cytometry analysis showed that obvious apoptosis occurred in TgTNFSF10-treated hemocytes and HepG2 cells. To sum up, our study demonstrated that TgTNFSF10 had strong apoptosis-inducing activity, which may participate in the innate immune response of T. granosa to pathogen invasion.

Functional characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) from blood clam Tegillarca granosa in innate immunity.

Lipopolysaccharide-induced TNF-alpha factor (LITAF), as a transcription factor, activates the transcription of TNF and other cytokines in inflammatory response upon lipopolysaccharide (LPS) stimulation. In the present study, we cloned and identified the full-length cDNA of LITAF homolog from blood clam Tegillarca granosa for the first time. The full-length cDNA of TgLITAF was 1801 bp encoding a polypeptide of 147 amino acids with an estimated molecular mass of 16.13 kDa. TgLITAF contained a zf-LITAF-like zinc ribbon domain at the C-terminal of the protein and the TgLITAF domain showed 48-74% amino acid sequence identity with other known LITAFs from other species. Subcellular localization study showed that TgLITAF was mainly expressed in the nucleus. qRT-PCR analysis showed that the TgLITAF transcription expressed constitutively in all the examined tissues with the highest expression level in the gills. After LPS or V. alginolyticus treatment, expression of TgLITAF in hemocytes was both up-regulated significantly at 3-6 h. Furthermore, in vitro study indicated that overexpression of TgLITAF in HeLa cells resulted in the activation of TNFα, p53, and influenced the expression levels of apoptotic-related genes Bax, Bcl-2, Caspase-3, Caspase-6, and Caspase-7. The proliferation of HeLa cells was inhibited by overexpression of TgLITAF. Apoptotic fluorescence assay further revealed that TgLITAF participated in the apoptotic process of HeLa cells. Western blotting analysis showed that overexpression of TgLITAF increased endogenous level of cleaved Caspase-7. Taken together, these results revealed that TgLITAF participates in the innate immune response to the pathogen invasion in blood clams and induces apoptosis in HeLa cells.

Immunotoxicity of four nanoparticles to a marine bivalve species, Tegillarca granosa.

The increasing application of nanomaterials drives the unintentional release of nanoparticles (NPs) into the ocean, which may pose a potential threat to marine organisms. It has been demonstrated that exposure to NPs could chanllenge the immune responses of marine species. However, the affecting mechanism behind remains poorly understood. In this study, the immunotoxic impacts and the mechanisms underpinning the effects of four major NPs, including nZnO, nFe2O3, nCuO, and carbon nanotube (MWCNT), were investigated in blood clam, Tegillarca granosa. The results showed that exposure to tested NPs resulted in reduced total counts, altered cell composition, and constrained phagocytic activities of haemocytes. The intracellular contents of reactive oxygen species (ROS) and the degree of DNA damage of haemocytes were significantly induced, whereas the haemocyte viability was suppressed. Furthermore, NP exposures led to significant increases in the in vivo contents of neurotransmitters. Down-regulations of the immune- and neurotransmitter-related genes were detected as well. Our data suggest that NP exposures hampered the immune responses of blood clams most likely through (1) inducing ROS, causing DNA damage, and reducing cell viability of haemocytes, (2) altering the in vivo contents of neurotransmitters, and (3) affecting the expression of immune- and neurotransmitter-related genes.

The synergic impacts of TiO2 nanoparticles and 17ß-estradiol (E2) on the immune responses, E2 accumulation, and expression of immune-related genes of the blood clam, Tegillarca granosa.

The extensive use of TiO2 nanoparticles (nTiO2) in industrial products has led to their release into the marine environment, thereby posing a potential risk to marine organisms. However, in addition to affecting marine organisms through its inherent properties, nTiO2 can also act as a vehicle for other toxic pollutants due to their strong adsorption ability through the "Trojan horse" effect. Due to their potential hazard, the endocrine disrupting chemicals (EDCs) such as 17ß-estradiol (E2), have been considered as one of the most serious anthropogenic threats to biodiversity and ecosystem health. However, there is still a lack of knowledge regarding the possible synergistic effects of nTiO2 and endocrine disrupting chemicals (EDCs) on marine organisms to date. Therefore, the combined effects of nTiO2 and 17ß-estradiol (E2) on the immune responses of the blood clam, Tegillarca granosa, were investigated in this study. After 10 days of treatment, the total number, phagocytic activity, red granulocytes ratio, and the phagocytosis of hemocytes were significantly reduced in almost all treatment groups. Furthermore, expressions of genes from NFκß and Toll-like receptor signaling pathways were significantly altered after exposure to nTiO2 and/or E2, indicating a reduced sensitivity to pathogen challenges. In addition, compared to exposure to E2 alone, co-exposure to E2 and nTiO2 led to a significant increase in the content of alkali-labile phosphate (ALP) in hemolymph, suggesting an enhanced E2 bioconcentration in the presence of nTiO2. In general, the present study demonstrated that nTiO2 enhanced the immunotoxicity of E2 to the blood clam, which may be due to the increased E2 uptake in the presence of nTiO2.

Molecular cloning, characterization and functional analysis of a putative mitogen-activated protein kinase kinase kinase 4 (MEKK4) from blood clam Tegillarca granosa.

The mitogen-activated protein kinase (MAPK) cascades stand for one of the most important signaling mechanisms in response to environmental stimuli. In the present study, we cloned and identified for the first time the full-length cDNA of MAPK kinase kinase 4 (TgMEKK4) from Blood clam Tegillarca granosa using rapid amplification of cDNA ends method. The full-length cDNA of TgMEKK4 was of 1605 bp in length, encoding a polypeptide of 364 amino acids with a predicted molecular mass of 41.22 kDa and theoretical isoelectric point of 6.29. The conserved MEKK4-domain was identified in TgMEKK4 by SMART program analysis. Homology analysis of the deduced amino acid sequence of TgMEKK4 with other known sequences revealed that TgMEKK4 shared 58%-80% identity to MEKK4s from other species. TgMEKK4 mRNA transcripts could be detected in all tissues examined with the highest expression level in the gill by qRT-PCR. The mRNA expression of TgMEKK4 was up-regulated significantly in hemocytes after Vibrio parahaemolyticus, Vibrio alginolyticus and Lipopolysaccharide (LPS) challenges. Overexpression of TgMEKK4 in HEK 293T cells resulted in the activation of JNK and ERK, but not p38. Consistently, In vivo study indicated that LPS stimulation enhanced JNK, ERK and p38 phosphorylation in blood clams. These results suggest that TgMEKK4 is a powerful factor in the regulation of genes that may be involved in innate immune response of blood clam.

Immunotoxicity of nanoparticle nTiO2 to a commercial marine bivalve species, Tegillarca granosa.

The increasing production and extensive application of nanoparticles (NPs) inevitably leads to increased release of NPs into the marine environment and therefore poses a potential threat to marine organisms, especially the sessile benthic bivalves. However, the impacts of NPs on the immunity of commercial and ecological important bivalve species, Tegillarca granosa, still remain unknown to date. In addition, the molecular mechanism of the immunotoxicity of NPs still remains unclear in marine invertebrates. Therefore, the immunotoxicity of nTiO2 exposure to T. granosa at environmental realistic concentrations was investigated in the present study. Results obtained showed that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were significantly reduced after 30 days nTiO2 exposures at the concentrations of 10 and 100 µg/L. Furthermore, the expressions of genes encoding Pattern Recognition Receptors (PPRs) and downstream immune-related molecules were significantly down-regulated by nTiO2 exposures, indicating a reduced sensitivity to pathogen challenges. In conclusion, evident immunotoxicity of nTiO2 to T. granosa at environmental realistic concentrations was detected by the present study. In addition, the gene expression analysis suggests that the PRRs (both TLRs and RIG1 investigated) may be the molecules for NPs recognition in marine invertebrates.

Gal/GalNAc specific multiple lectins in marine bivalve Anadara granosa.

Complete lectin mapping of molluscs with their diversified recognition pattern and possible role in lectin-carbohydrate interaction based immune response triggering need much attention. In this communication, Gal/GalNAc specific three lectins AGL-IA (Anadara granosa lectin-IA), AGL-IB (A. granosa lectin-IB) and AGL-IV (A. granosa lectin-IV) and a lectin having hemolytic activity AGL-III (A. granosa lectin-III) were purified from the plasma of A. granosa bivalve by a combination of gel filtration and affinity chromatography. AGL-IA and IB were oligomeric lectins whereas, AGL-III and IV were monomeric. The molecular weight of AGL-IA, IB, III and IV were 375, 260, 45 and 33 kDa respectively. AGL-IA and IV agglutinated both rabbit and pronase treated human erythrocytes, whereas AGL-IB agglutinated only rabbit erythrocytes. AGL-III was found to agglutinate rabbit erythrocytes, however, it caused hemolysis of pronase treated human erythrocytes. The activity of all four lectins was calcium dependent and maximum at a pH range 7-8. Apart from Gal/GalNAc specific, the four lectins showed substantial differences in their carbohydrate recognition pattern. Moreover, there was a difference in the carbohydrate specificity between AGL-III and other three lectins (AGL-IA, AGL-IB and AGL-IV) towards polyvalent glycotope. On the one hand, 'cluster glycoside effect' i.e., an enhancement of the activity of a multivalent ligand, was observed for carbohydrate specificities of AGL-IA, AGL-IB, AGL-IV. On the other hand, the effect of multivalent ligands on the carbohydrate specificity of AGL-III was opposite of cluster glycoside effect. The affinity of AGL-IA, AGL-IB and AGL-IV for ligands can be ranked as follows: glycoproteins >> polysaccharide > oligosaccharides and monosaccharides. However, Gal related monosaccharides were the best inhibitors of AGL-III and the inhibitory activity decreased gradually in the following order: monosaccharide > disaccharide > polysaccharide. Thus, the diverse specificity of multiple lectins in A. granosa plasma possibly enables to recognize a wide range of microorganisms.

Polymorphism of the multiple hemoglobins in blood clam Tegillarca granosa and its association with disease resistance to Vibrio parahaemolyticus.

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, but it can serve additional functions beyond the transport of oxygen. In this study, we identified polymorphism in the blood clam Tegillarca granosa Hb (Tg-Hb) genes and investigated the association of this polymorphism with resistance/susceptibility to Vibrio parahaemolyticus. Analysis of the 540 sequences revealed 28 SNPs in the coding region of three Tg-Hbs, corresponding to about one SNP per 48 bp. Three SNPS: HbIIA-E2-146, HbIIB-E2-23, HbIIB-E2-121 showed a significant association with resistance/susceptibility to V. parahaemolyticus (P < 0.05). To further demonstrate that three significant SNPs of Tg-Hbs is associated with resistance of clams to V. parahaemolyticus, SNPs were genotyped in V. parahaemolyticus resistant strain clams and the wild base population from which this strain was derived. The results indicated that the nonsynonymous mutation T allele at HbIIA-E2-146 and A allele at HbIIB-E2-23 are associated with V. parahaemolyticus resistance in the blood clam, and its association with disease resistance may be due to its cause changes in amino acid sequences to a functional polymorphism. Together with previous bacterial challenge study, these results provides direct evidence that variation at HbIIA-E2-146 and HbIIB-E2-23 are associated with disease resistance in the blood clam, and these two polymorphic loci could be potential gene markers for the future molecular selection of strains that are resistant to diseases caused by V. parahaemolyticus.

A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis.

Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys³° responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of Tg-TIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4-like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms.

Hemoglobin of the bloody clam Tegillarca granosa (Tg-HbI) is involved in the immune response against bacterial infection.

Hemoglobins (Hb) are the major protein components of erythrocytes circulating in the red blood, but can serve additional functions besides the transport of oxygen. Here, the cDNA of the bloody clam (Tegillarca granosa) Hb dimer (designated Tg-HbI) was cloned and was found to be 748 bp in length, consisting of an open reading frame of 441 bp encoding a polypeptide of 147 amino acids. The deduced amino acid sequence of Tg-HbI shared 81.6% similarity with HbI from two species of the genus Scapharca and 46-51% similarity with the Hb proteins from other mollusks. The 3D structure of bloody clam Tg-HbI was predicted by the SWISS-MODEL Protein Modelling Server and compared with that of Scapharca kagoshimensis. The mRNA transcript of Tg-HbI was detected in all of the clam cells/tissues examined, including haemocytes, the adductor muscle, foot, hepatopancreas, gill and mantle. The mRNA expression of Tg-HbI was significantly up-regulated after Vibrio parahaemolyticus, lipopolysaccharide and peptidoglycan challenge, indicating that Tg-HbI was involved in the immune defence responses against bacterial infection and exposure to bacterial pathogenic factors. As the first functional research on the Hb protein in bloody clam, our findings provide new insight into the innate immune defence mechanisms of T. granosa and other mollusks.