An ancient C-type lectin in Chlamys farreri (CfLec-2) that mediate pathogen recognition and cellular adhesion

C-type lectins are a superfamily of Ca2+ dependent carbohydrate-recognition proteins which play significant diverse roles in nonself-recognition and clearance of invaders. In the present study, a C-type lectin (CfLec-2) from Zhikong scallop Chlamys farreri was selected to investigate its functions in innate immunity. The mRNA expression of CfLec-2 in hemocytes was significantly up-regulated (P < 0.01) after scallops were stimulated by LPS. PGN or beta-glucan, and reached the highest expression level at 12h post-stimulation, which was 72.5-, 23.6- or 43.8-fold compared with blank group, respectively. The recombinant Cflec-2 (designated as rCfLec-2) could bind LPS, PGN, mannan and zymosan in vitro, but it could not bind beta-glucan. Immunofluorescence assay with polyclonal antibody specific for Cflec-2 revealed that CfLec-2 was mainly located in the mantle, kidney and gonad. Furthermore, rCfLec-2 could bind to the surface of scallop hemocytes, and then initiated cellular adhesion and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-2 serum. These results collectively suggested that CfLec-2 from the primitive deuterostome C. farreri could perform two distinct immune functions, pathogen recognition and cellular adhesion synchronously, while these functions were performed by collectins and selectins in vertebrates, respectively. The synchronous functions of pathogen recognition and cellular adhesion performed by CfLec-2 tempted us to suspect that CfLec-2 was an ancient form of C-type lectin, and apparently the differentiation of these two functions mediated by C-type lectins occurred after mollusk in phylogeny. (C) 2010 Elsevier Ltd. All rights reserved.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens

Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. (C) 2010 Elsevier Ltd. All rights reserved.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NF kappa B is a family of transcription factors. In the present study, a Rel/NF kappa B family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW)of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical pl was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NF kappa B member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of infection of EGH-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for Immoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage I h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians. (c) 2005 Elsevier B.V. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at http://www.penbase.immunaqua.com, has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature. (c) 2005 Elsevier Ltd. All rights reserved.

Peptidoglycan recognition protein of Chlamys farreri (CfPGRP-S1) mediates immune defenses against bacterial infection

Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in detecting and eliminating the invading bacteria. Several PGRPs have been identified from mollusk, but their functions and the underlined mechanism are still unclear. In the present study, the mRNA expression profiles, location, and possible functions of PGRP-S1 from Zhikong scallop Chlamys farreri (CfPG RP-St) were analyzed. The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation (P < 0.01), while moderately after the stimulations of LPS (P < 0.01) and beta-glucan (P < 0.05). The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bind beta-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+. These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved