A novel invertebrate toll-like receptor with broad recognition spectrum from thick shell mussel Mytilus coruscus.

Toll-like receptors (TLRs) are a category of most well recognized pattern recognition molecules that act on a vital role in both innate and adaptive immunity. In the present study, a novel toll-like receptor (McTLRw) was identified and characterized in thick shell mussel Mytilus coruscus. McTLRw possesses one intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain, one transmembrane region (TM), one leucine rich repeat N-terminal domain (LRR_NT) and a few of leucine-rich repeats (LRRs), which all are common in typical TLRs. McTLRw transcripts were constitutively expressed in all examined tissues with higher expression levels in immune related tissues, and were significantly induced in haemocytes with the challenges of live Vibrio alginolyticus, lipopolysaccharide (LPS), peptidoglycans (PGN) and ß-glucan (GLU), but not induced by polyinosinic-polycytidylic acid (poly I:C). rMcTLRw exhibited affinity to LPS, PGN and GLU while no affinity to poly I:C. Further, the downstream of TLR signaling pathway myeloid differentiation factor 88a (MyD88a), interleukin-1 receptor-associated kinase-4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were significantly repressed in McTLRw silenced mussels while challenged with LPS. These results collectively indicated that McTLRw is one member of TLR family and involved in immune response to against invaders by taking participate in TLR mediated signaling pathway.

Immunity in mussels: An overview of molecular components and mechanisms with a focus on the functional defenses.

Bivalves' immunity has received much more attention in the last decade, which resulted to a valuable growth in the availability of its molecular components. Such data availability coupled with the economical importance of these organisms aimed to shift the increase in the number of immunological and stress-related studies. Unfortunately, the crowd of generated data deciphering the involved physiological processes, investigators' differential conceptualization and the aimed objectives, has complicated the sensu stricto outlining of immune-related mechanisms. Overall, this review tried to compiles a summary about the molecular components of the mussels' immune response, surveying an overview of the mussels' functional immunity through gathering the most recent-related topics of bivalves' immunity as apoptosis and autophagy which deserves a great attention as stress-related mechanisms, the disseminated neoplasia as outbreak transmissible disease, not only within the same specie but also among different species, the hematopoiesis as topic that still generating interesting debate in the scientific community, the mucosal immunity described as the interface where host-pathogen interactions would occurs and determinate the late immune response, and innate immune memory and transgenerational priming, which described as very recent research topic with extensive applications in shellfish farming industry.

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in thick shell mussel, Mytilus coruscus.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Despite of the well study in vertebrates, there is few data ascribe to this TRAF member in invertebrates, especially in bivalves. In the present study, a novel TRAF6 homologue termed McTRAF6 was firstly characterized in Mytilus coruscus. Like its counterparts in mammals, McTRAF6 shared the domain topology containing one RING domain, two zinc finger domains, one coiled-coil region and a MATH domain. McTRAF6 transcripts predominantly expressed in gills, digestive glands and hemocytes in M. coruscus, and were significantly up-regulated in hemocytes after challenge with lipopolysaccharide (LPS) and polyinosine-polycytidylic acid (poly I:C). Further, the subcellular localization in cytoplasm and the activation of Nk-κB or ISRE luciferase reporter by overexpressed McTRAF6 were identified in HEK293T cells. These results collectively indicate that McTRAF6 is a member of TRAF6 subfamily and plays a potential role in immune defense system against pathogenic agents invasions in thick shell mussel. To our knowledge, this is the first report on component of TLR signaling pathway in thick shell mussel, providing further evidence for the existence of TLR pathway in M. coruscus and contribute to clarify the innate immune system of thick shell mussel.

cDNA and Gene Structure of MytiLec-1, A Bacteriostatic R-Type Lectin from the Mediterranean Mussel (Mytilus galloprovincialis).

MytiLec is an α-d-galactose-binding lectin with a unique primary structure isolated from the Mediterranean mussel (Mytilus galloprovincialis). The lectin adopts a ß-trefoil fold that is also found in the B-sub-unit of ricin and other ricin-type (R-type) lectins. We are introducing MytiLec(-1) and its two variants (MytiLec-2 and -3), which both possess an additional pore-forming aerolysin-like domain, as members of a novel multi-genic "mytilectin family" in bivalve mollusks. Based on the full length mRNA sequence (911 bps), it was possible to elucidate the coding sequence of MytiLec-1, which displays an extended open reading frame (ORF) at the 5' end of the sequence, confirmed both at the mRNA and at the genomic DNA sequence level. While this extension could potentially produce a polypeptide significantly longer than previously reported, this has not been confirmed yet at the protein level. MytiLec-1 was revealed to be encoded by a gene consisting of two exons and a single intron. The first exon comprised the 5'UTR and the initial ATG codon and it was possible to detect a putative promoter region immediately ahead of the transcription start site in the MytiLec-1 genomic locus. The remaining part of the MytiLec-1 coding sequence (including the three sub-domains, the 3'UTR and the poly-A signal) was included in the second exon. The bacteriostatic activity of MytiLec-1 was determined by the agglutination of both Gram-positive and Gram-negative bacteria, which was reversed by the co-presence of α-galactoside. Altogether, these data support the classification of MytiLec-1 as a member of the novel mytilectin family and suggest that this lectin may play an important role as a pattern recognition receptor in the innate immunity of mussels.

The response profiles of HSPA12A and TCTP from Mytilus galloprovincialis to pathogen and cadmium challenge.

Heat shock 70 kDa protein 12A (HSPA12A) is an atypical member of HSP70 family, and the translationally controlled tumor protein (TCTP) is a novel HSP with chaperone-like activity. They are both involved in protecting organisms against various stressors. In the present study, the cDNAs of HSPA12A and TCTP (called MgHSPA12A and MgTCTP) were identified from Mytilus galloprovincialis by RACE approaches. The full-length cDNA of MgHSPA12A and MgTCTP encoded a peptide of 491 and 171 amino acids, respectively. Real-time PCR was employed to analyze the tissue distribution and temporal expression of these two genes after bacterial challenge and cadmium (Cd) exposure. It was found that the transcripts of MgHSPA12A and MgTCTP were dominantly expressed in gonad and muscle, respectively. The expression level of MgTCTP at 48 h post Vibrio anguillarum challenge was detected to be significantly up-regulated in hepatopancreas (P < 0.05). As concerned to Cd exposure, 2.0-fold increase of MgHSPA12A expression compared to that of the control was observed at 48 h in 5 µg/L Cd(2+)-treated group, while the expression levels of MgTCTP were significantly decreased after exposed to both 5 and 50 µg/L Cd(2+) for 24 h and 96 h. These results suggested the potential involvement of MgHSPA12A and MgTCTP in the mediation of the immune responses and environmental stress in mussels.

Molecular characterization of a manganese superoxide dismutase and copper/zinc superoxide dismutase from the mussel Mytilus galloprovincialis.

The full-length cDNA sequences coding respectively for a manganese superoxide dismutase (Mg-MnSOD) and copper/zinc superoxide dismutase (Mg-CuZnSOD) were cloned from Mytilus galloprovincialis. Mg-MnSOD and Mg-CuZnSOD cDNAs encoded a polypeptide of 228 and 211 amino acids, respectively. Sequence analysis indicated Mg-MnSOD was a mitochondrial MnSOD and Mg-CuZnSOD was an intracellular CuZnSOD. Multiple alignment analysis showed that both Mg-MnSOD and Mg-CuZnSOD sequences had the common features conserved in MnSODs and CuZnSODs, respectively. Phylogenetic analysis revealed that Mg-MnSOD clustered together with MnSODs from other mollusks, whereas Mg-CuZnSOD clustered with other mollusk intracellular CuZnSODs with a wider phylogenetic distance. By quantitative real-time RT-PCR (qPCR) analysis, both Mg-MnSOD and Mg-CuZnSOD transcripts were detected in all tissues examined with the highest expression level in hepatopancreas. Following bacterial challenge, the expression level of Mg-MnSOD and Mg-CuZnSOD increased first and subsequently decreased to the original level in hemocytes. In hepatopancreas, Mg-CuZnSOD mRNA was up-regulated significantly at 72 h and 96 h post challenge, while the level of Mg-MnSOD transcript had no significant change. Therefore, Mg-MnSOD and Mg-CuZnSOD expressions were inducible and they were probably involved in the immune response against bacterial challenge. These results suggest that these SODs may play important roles in the immune defense system of M. galloprovincialis and perhaps contribute to the protective effects against oxidative stress in this mussel.

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signaling pathway in Lophotrochozoa.

Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus.

Antimicrobial peptides (AMPs) are components of the innate immune responses that form the first line of host defense against pathogens. Marine mussels can produce a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes. In the current study, we purified and characterized a novel cysteine-rich peptide with remarkable antibacterial activity from Mytilus coruscus and designated with myticusin-1, a 104-amino acid long polypeptide including 10 cysteine residues forming an unusual cysteine pattern. Antimicrobial assays demonstrated that myticusin-1 exhibited stronger anti-microbial properties against Gram-positive bacteria more than Gram-negative bacteria and fungus. Furthermore, myticusin-1 caused significant morphological alterations in both Sarcina luteus and Escherichia coli as shown by transmission electron microscopy (TEM). The cDNA of myticusin-1 was cloned and sequenced from the hemocytes cDNA library of M. coruscus. The mRNA transcripts of myticusin-1 are mainly detected in hemocyte, which indicates that myticusin-1 are specifically synthesized and stored in circulating hemocytes. The expression level of myticusin-1 in hemocytes was up-regulated and reached the highest level at 36 h after S. luteus challenge, which was 20-fold increase compared to that of the control group. These results indicated that myticusin-1 was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

Involvement of pore-forming molecules in immune defense and development of the Mediterranean mussel (Mytilus galloprovincialis).

The membrane attack complex and perforin (MACPF) superfamily is one of the largest families of pore-forming molecules. Although MACPF proteins are able to destruct invading microbes, several MACPF proteins play roles in embryonic development, neural migration or tumor suppression. We describe two apextrin-like proteins (ApelB and ApelP) and one MACPF-domain-containing protein (Macp) in Mytilus galloprovincialis. The two apextrin-like proteins did not present any conserved domain. The Macp protein contained the membrane/attack complex domain and its signature motif. Gene expression during larval development was analyzed by RT-PCR. There was a stage-specific up-regulation of the three proteins, suggesting that they play a role in development. Apextrin-like proteins were highly expressed at blastula and trochophore stage, whereas Macp was expressed at veliger stage. RT-PCR revealed up-regulation of the three genes in tissues and hemocytes from adults treated with bacteria and pathogen-associated molecular patterns, suggesting that they may be involved in the immune response.

Effect of 17ß-estradiol on adhesion of Mytilus galloprovincialis hemocytes to selected substrates. Role of alpha2 integrin subunit.

The process of hemocyte adhesion to extracellular matrix (ECM) proteins plays a crucial role in cell immunity. In most of these interactions between ECM proteins and cells, integrins are involved. The results of the present study showed that incubation of Mytilus galloprovincialis hemocytes with 17ß-estradiol caused significant increased adhesion of hemocytes to ECM proteins and specifically to laminin-1, collagen IV and oxidized collagen IV, in relation to control cells. The adhesion of hemocytes to oxidized collagen was significantly higher than to either collagen IV or to laminin-1. In accordance with this, inhibition of either NADPH oxidase or nitric oxide (NO) synthase attenuated 17ß-estradiol effect on hemocyte adhesion, suggesting that the high levels of free radicals, produced after 17ß-estradiol effect, could contribute to the high adhesion of hemocytes to laminin-1 and collagen IV. The implication of ROS was further confirmed by the use of the oxidant rotenone, which caused elevation of cell adhesion in relation to control and by the antioxidant NAC which attenuated 17ß-estradiol effect. The mechanism of 17ß-estradiol induced adhesion to laminin-1, collagen IV and oxidized collagen IV involves a large number of intracellular components, as Na+/H+ exchanger (NHE), all isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK) as well as alpha2 integrin subunit. Maintenance of high cyclic adenosine-3'-5'-monophosphate (cAMP) levels caused non significant higher adhesion of hemocytes to ECM proteins in relation to control cells. Our results showed that 17ß-estradiol caused a significant increase in α2 integrin subunit levels, which was reduced after inhibition of NHE, PI3K, PKC, NO synthase, NADPH oxidase and JNK. In addition, our results showed that apart from 17ß-estradiol, high cAMP and high ROS levels caused significantly higher induction of α2 integrin subunit levels in relation to control. Our results imply a potential involvement of cAMP in immune responses of Mytilus hemocytes, which needs further investigation.

Influence of beta-glucans on the immune responses of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis).

The effects of beta-glucans on several immune functions of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis) hemocytes were determined. Nitric oxide (NO) production increased significantly in beta-glucan treated mussels and clams. In mussels, beta-glucans increased by themselves the release of free oxygen radicals and also were able to enhance the phorbol 12-myristate 13-acetate (PMA) mediated effect on this hemocyte activity. However, high doses of beta-glucans when combined with zymosan decreased this respiratory burst. In clams, hemolymph treated with several doses of beta-glucans limited the growth of the three bacteria, Vibrio algynolyticus (strain TA15), Vibrio splendidus (strain TA2) and Escherichia coli (strain ATCC 13706). This modulation on the antibacterial activity, however, was not observed when mussel hemolymph was incubated with beta-glucans. These results suggest that the immune responses of these animals can be up and down modulated by external stimuli and, although clams and mussels are both relatively closely related species, their behaviour concerning immune responses can be different.

High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions.

Small cationic antimicrobial peptides (AMPs) are host defense molecules detected in virtually all groups of organisms. To investigate the immune response mechanisms of Mytilus galloprovincialis, primary and suppression subtractive hybridization libraries were prepared from hemolymph of mussels injected with heat-inactivated bacteria or poly I:C, the latter mimicking viral infection. After DNA sequencing, sequence processing and similarity searching, a remarkable abundance of AMP mRNAs were identified. In detail, 25.9% and 32.4% AMP sequences from mussels infected with bacteria and 43.4% and 40.6% from mussels stimulated with poly I:C were detected by selective amplification of 180 differentially expressed genes and random sequencing of 967 cDNA clones, respectively. The 232 ESTs matching with myticin A and B (Mytilus spp.) displayed considerable sequence variability and revealed a third cluster proposed here as myticin C. Phenetic analysis of the translated myticin ESTs yielded 74 and 25 variants of the precursor and active peptide, respectively, and confirmed the high polymorphism of the new form. Myticin C shows typical features of the CSalphabeta AMP family (eight-cysteine array and secretory signal peptide) as well as amino acid variation, mainly in the anionic C-terminal region. The sequencing of one intronic region from genomic DNA, allowed us to detect 13 variants in 9 individual mussels referring them to one gene only. In addition to hemolymph, myticin C transcripts were detected in various mussel tissues, oocytes and early larval stages. The striking sequence variability and expression levels of myticins in mussels confirm the fundamental role of these natural antibiotics in the ancient host-pathogen interplay of mutual inhibition, evasion and adaptation strategies.

After the Prestige oil spill modifications in NO production and other parameters related to the immune response were detected in hemocytes of Mytilus galloprovincialis.

In marine mollusks, many physiologic functions are regulated seasonally depending on such factors as the reproductive cycle or the presence of food. The synthesis of nitric oxide by hemocytes of Mytilus galloprovincialis is among the multiple physiologic actions in the immune response, and it is also affected by season. The maximal basal production of NO by hemocytes of M. galloprovincialis was detected in summer, whereas the minimum values were detected in winter. In winter, the presence of IL-2 induced an increase in NO production that was not detected in summer. Three months after the Prestige oil spill (November 2002), basal NO production by the hemocytes of mussels in the Galician coast showed a progressive decrease and stopping, both in summer and in winter. The characteristic increase of NO synthesis induced by IL-2 in winter also disappeared all through 2003 and 2004. The two different nitric oxide synthases previously identified by immunoblotting between 1999 and 2002 were undetectable in both 2003 and 2004. When comparing the data obtained during 2003 and 2004 to those obtained in previous years, an increase in the proportion of SH cells was detected. Also, these cells showed a higher sensitivity to apoptosis- and necrosis-inducing agents than in earlier years.

Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies.

Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17alpha-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.

Assessment of in vivo effects of the prestige fuel oil spill on the mediterranean mussel immune system.

A laboratory experiment was carried out to study immune function alteration of the mussel Mytilus galloprovincialis when exposed to the Prestige oil spilled in November 2002 on the northwestern Spanish coast. Mussels were maintained for 4 months in tanks with flowing seawater and with 1, 2, and 0 kg (controls) Prestige fuel oil. Polycyclic aromatic hydrocarbon concentrations, which were determined in gills and digestive glands, were higher in digestive glands. The methylphenantrene and dibenzothiophene profiles confirmed the real exposure of mussels to the fuel oil. Immune data analysis revealed that no differences between fuel-treated and control animals were found in the cellular immune parameters measured (hemocyte viability, phagocytic activity, nitric oxide production, and chemiluminescence emission). In addition, histologic observations did not reveal tissue lesions in any of the samples, probably because of the short time of fuel-oil exposure. In contrast, significant differences were found in serum protein concentration and lysozyme activity between the fuel-treated mussels and controls. However, these humoral immune parameters were dependant on numerous environmental and physiologic factors, so it was difficult to ascertain the real effect of the fuel oil on their variability. Because hemocytes are the primary line of defense of bivalve mollusks, the results obtained in the present study suggest that the mussel immune system was not significantly affected by exposure to the Prestige fuel oil.

Immunomodulation by 17beta-estradiol in bivalve hemocytes.

In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17beta-estradiol (E(2)) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E(2) in Mytilus hemocytes, the cells responsible for the innate immune response. E(2) at 5-25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E(2) inhibited phagocytosis. E(2)-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine and superoxide dismutase, indicating involvement of NO and O(2)(-); NO production was confirmed by nitrite accumulation. The effects of E(2) were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E(2) induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-alpha- and -beta-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E(2) on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E(2)-injected mussels. E(2) significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E(2) resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E(2) in immunomodulation is conserved from invertebrates to mammals.