Big Defensin Research Articles

The immune response of the scallop Argopecten purpuratus is associated with changes in the host microbiota structure and diversity

All organisms live in close association with a variety of microorganisms called microbiota. Furthermore, several studies support a fundamental role of the microbiota on the host health and homeostasis. In this context, the aim of this work was to determine the structure and diversity of the microbiota associated with the scallop Argopecten purpuratus, and to assess changes in community composition and diversity during the host immune response. To do this, adult scallops were immune challenged and sampled after 24 and 48 h. Activation of the immune response was established by transcript overexpression of several scallop immune response genes in hemocytes and gills, and confirmed by protein detection of the antimicrobial peptide big defensin in gills of Vibrio-injected scallops at 24 h post-challenge. Then, the major bacterial community profile present in individual scallops was assessed by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes and dendrogram analyses, which indicated a clear clade differentiation of the bacterial communities noticeable at 48 h post-challenge. Finally, the microbiota structure and diversity from pools of scallops were characterized using 16S deep amplicon sequencing. The results revealed an overall modulation of the microbiota abundance and diversity according to scallop immune status, allowing for prediction of some changes in the functional potential of the microbial community. Overall, the present study showed that changes in the structure and diversity of bacterial communities associated with the scallop A. purpuratus are detected after the activation of the host immune response. Now, the relevance of microbial balance disruption in the immune capacity of the scallop remains to be elucidated.

Reproduction Immunity Trade-Off in a Mollusk: Hemocyte Energy Metabolism Underlies Cellular and Molecular Immune Responses.

Immune responses, as well as reproduction, are energy-hungry processes, particularly in broadcast spawners such as scallops. Thus, we aimed to explore the potential reproduction-immunity trade-off in Argopecten purpuratus, a species with great economic importance for Chile and Peru. Hemocytes, key immunological cells in mollusks, were the center of this study, where we addressed for the first time the relation between reproductive stage, hemocyte metabolic energetics and their capacity to support immune responses at cellular and molecular levels. Hemocyte metabolic capacity was assessed by their respiration rates, mitochondrial membrane potential and citrate synthase (CS) activity. Cellular immune parameters such as the number of circulating and tissue-infiltrating hemocytes and their reactive oxygen species (ROS) production capacity were considered. Molecular immune responses were examined through the transcriptional levels of two pattern recognition receptors (ApCLec and ApTLR) and two anti-microbial effectors (ferritin and big defensin). Their expressions were measured in hemocytes from immature, matured and spawned scallops under basal, and one of the following challenges: (i) in vitro, where hemocytes were challenged with the β glucan zymosan, to determine the immune potentiality under standardized conditions; or (ii) in vivo challenge, using hemocytes from scallops injected with the pathogenic bacteria Vibrio splendidus. Results indicate a post-spawning decrease in the structural components of the immune system (hemocyte number/quality) and their potential capacity of performing immune functions (with reduced ATP-producing machinery and exhaustion of energy reserves). Both in vitro and in vivo challenges demonstrate that hemocytes from immature scallops have, in most cases, the best metabolic potential (increased CS activity) and immune performances, with for example, over threefold higher ROS production and tissue-infiltration capacity than those from mature and spawned scallops after the bacterial challenge. Agreeing with cellular responses, hemocytes from immature individuals induced the highest levels of immune receptors and antimicrobial effectors after the bacterial challenge, while spawned scallops presented the lowest values. Overall, results suggest a trade-off between resource allocation in reproduction and the immune responses in A. purpuratus, with hemocyte energy metabolic capacity potentially underlying cellular and molecular immune responses. Further research would be necessary to explore regulatory mechanisms such as signaling pleiotropy which may potentially be underlying this trade-off.

Molecular characterization and protein localization of the antimicrobial peptide big defensin from the scallop Argopecten purpuratus after Vibrio splendidus challenge

Big defensins are antimicrobial peptides (AMPs) that are proposed as important effectors of the immune response in mollusks, chelicerates and chordates. At present, only two members of the big defensin family have been identified in scallop. In the present work, a cDNA sequence encoding a new big defensin homologue was characterized from the scallop Argopecten purpuratus, namely ApBD1. ApBD1 cDNA sequence comprised 585 nucleotides, with an open reading frame of 375 bp and 5'- and 3′-UTRs of 41 and 167 bp, respectively. The deduced protein sequence contains 124 amino acids with a molecular weight of 13.5 kDa, showing characteristic motifs of the big defensin family and presenting 76% identity with the big defensin from the scallop A. irradians. Phylogenetic analysis revealed that ApBD1 is included into the cluster of big defensins from mollusks. Tissue-specific transcript expression analysis by RT-qPCR showed that ApBD1 was present in all tissues tested from non-immune challenged scallops but it was most strongly expressed in the mantle. The transcript levels of ApBD1 were significantly up-regulated in gills at 24 and 48 h post-injection with the heat-attenuated bacteria Vibrio splendidus. Additionally, immunofluorescence analysis using a polyclonal anti-ApBD1 antibody showed that this protein was abundantly located in epithelial linings of gills and mantle; and also in digestive gland showing ApBD1-infiltrating hemocytes from immune challenged scallops. This is the first time that a big defensin is detected and located at the protein level in a mollusk. These results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus.

MicroRNA expression changes after lipopolysaccharide treatment in gills of amphioxus Branchiostoma belcheri

Recently, amphioxus has served as a model for studying the origin and evolution of vertebrate immunity. However, little is known about how microRNAs (miRNAs) are involved in the immune defense in amphioxus. In this article, we identified the amphioxus miRNAs in the acute-phase response to lipopolysaccharide (LPS). We determined the time point for the peak of immune response in amphioxus after LPS challenge by evaluating the expression of Branchiostoma belcheri toll-like receptor 1, NF-κb (c-Rel), and big defensin which react with pathogen-associated molecular patterns(PAMPs). Then we chose 12 h as the point to perform miRNA microarray analysis to select the differentially expressed miRNAs. Furthermore, we used quantitative real-time PCR to detect the expression patterns of selected amphioxus miRNAs under effective LPS challenge during the time course. The microarray data revealed that the miRNA expression file was significantly changed after LPS stimulation. The changes of the 10 most upregulated and 7 most downregualted miRNAs in gills of the amphioxus following challenge with LPS revealed a temporal induction kinetic. Our current study will provide valuable information to take an insight into molecular mechanism of innate immune and the evolution of the miRNA family.

Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopecten purpuratus

Reactive oxygen intermediates (ROI) are metabolites produced by aerobic cells which have been linked to oxidative stress. Evidence reported in vertebrates indicates that ROI can also act as messengers in a variety of cellular signaling pathways, including those involved in innate immunity. In a recent study, an inhibitor of NF-kB transcription factors was identified in the scallop Argopecten purpuratus, and its functional characterization suggested that it may regulate the expression of the big defensin antimicrobial peptide ApBD1. In order to give new insights into the messenger role of ROI in the immune response of bivalve mollusks, the effect of ROI production on gene transcription of ApBD1 was assessed in A. purpuratus. The results showed that 48 h-cultured hemocytes were able to display phagocytic activity and ROI production in response to the β-glucan zymosan. The immune stimulation also induced the transcription of ApBD1, which was upregulated in cultured hemocytes. After neutralizing the ROI produced by the stimulated hemocytes with the antioxidant trolox, the transcription of ApBD1 was reduced near to base levels. The results suggest a potential messenger role of intracellular ROI on the regulation of ApBD1 transcription during the immune response of scallops.

Cloning of a big defensin gene and its response to Vibrio parahaemolyticus challenge in the noble scallop Chlamys nobilis (Bivalve: Pectinidae).

The noble scallop Chlamys nobilis has been an important marine cultured bivalve in the Southern Sea of China for decades. However, large-scale mortality events often occurred during the scallop' cultivation. As one of AMPs (antimicrobial peptides), big defensin is an important component of the innate immunity against pathogenic microorganisms in invertebrates. In order to investigate whether the big defensin can play a role in the immune defense against pathogenic microorganisms in noble scallop, a big defensin gene from the hemocytes of Chlamys nobilis (CnBD) was cloned, and the mRNA level was measured after an acute Vibrio parahaemolyticus challenge of 36h. The CnBD cDNA contains an open reading frame (ORF) of 381bp encoding a peptide of 126 amino acids residues. The deduce amino acid sequence of CnBD shows a high similarity with that from Argopecten irradians and displays common features of big defensin, indicating that CnBD is a new member of the big defensin family. Compared with the control group, the relative mRNA level of CnBD was significantly up-regulated at 3, 24 and 36h. The present result indicated that CnBD played an immune role against bacterial infection in noble scallop.

Reactive oxygen intermediates promotes the expression of an antimicrobial peptide in the northern scallop Argopecten purpuratus hemocytes in vitro

Reactive oxygen intermediates are metabolites normally produced on aerobic metabolisms and has been linked to processes like oxidative stress and aging. Despite this negative paradigm about ROI, another set of evidence in mammals suggest that these reactive intermediates can act as important second messengers in a variety of cellular signaling processes such as those involved on the immune response. Primary cultures of hemocytes from the Chilean scallop Argopecten purpuratus were developed to study a possible link between the production of reactive oxygen intermediates and the expression of an antimicrobial peptide, big defensin, under a context of immune response. A modified Leibovitz L-15 medium supplemented with 50% of sterile plasma from scallop was essential for hemocyte adhesion an survival. The ability of these primary cultures to develop an immune response was confirmed by phagocytic activity and ROI production essays. The antimicrobial peptide big defensin was up-regulated in response to the β-glucan zymosan and this up regulation could be diminished near to constitutive levels by the treatment of cultured hemocytes with the antioxidant trolox for 3 hours before the immune induction. This results constitutes the first evidence on invertebrates that supports a rol of intracellular oxygen intermediates as promoters of the expression of an antimicrobial effector during an immune response.

Molecular characterization of an inhibitor of NF-κB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk.

Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates. The activation of the Rel/NF-κB signaling pathway depends largely in the degradation of IκB proteins and thus, IκBs are a main target for the identification of genes whose expression is controlled by Rel/NF-κB pathway. In order to identify such regulation in bivalve mollusks, the cDNA sequence encoding an IκB protein was characterized in the scallop Argopecten purpuratus, ApIκB. The cDNA sequence of ApIκB is comprised of 1480 nucleotides with a 1086 bp open reading frame encoding for 362 amino acids. Bioinformatics analysis showed that ApIκB displays the conserved features of IκB proteins. The deduced amino acid sequence consists of a 39.7 kDa protein, which has an N-terminal degradation motif, six ankyrin repeats and a C-terminal phosphorylation site motif. Phylogenetic analysis revealed a high degree of identity between ApIκB and other IκBs from mollusks, but also to arthropod cactus proteins and vertebrate IκBs. Tissue expression analysis indicated that ApIκB is expressed in all examined tissues and it is upregulated in circulating hemocytes from scallops challenged with the pathogenic Gram-negative bacterium Vibrio splendidus. After inhibiting ApIκB gene expression using the RNA interference technology, the gene expression of the antimicrobial peptide big defensin was upregulated in hemocytes from non-challenged scallops. Results suggest that ApIκB may control the expression of antimicrobial effectors such as big defensin via a putative Rel/NF-κB signaling pathway. This first evidence will help to deepen the knowledge of the Rel/NF-κB conserved pathway in scallops.

High polymorphism in big defensin gene expression reveals presence–absence gene variability (PAV) in the oyster Crassostrea gigas

We report here the first evidence in an invertebrate, the oyster Crassostrea gigas, of a phenomenon of Presence–Absence Variation (PAV) affecting immune-related genes. We previously evidenced an extraordinary interindividual variability in the basal mRNA abundances of oyster immune genes including those coding for a family of antimicrobial peptides, the big defensins (Cg-BigDef). Cg-BigDef is a diverse family composed of three members: Cg-BigDef1 to -3. Here, we show that besides a high polymorphism in Cg-BigDef mRNA expression, not all individual oysters express simultaneously the three Cg-BigDefs. Moreover, in numerous individuals, no expression of Cg-BigDefs could be detected. Further investigation at the genomic level revealed that in individuals in which the transcription of one or all Cg-BigDefs was absent the corresponding Cg-bigdef gene was missing. In our experiments, no correlation was found between Cg-bigdef PAV and oyster capacity to survive Vibrio infections. The discovery of P-A immune genes in oysters leads to reconsider the role that the immune system plays in the individual adaptation to survive environmental, biotic and abiotic stresses.

Simultaneous and Systemic Knock-down of Big Defensin 1 and 2 gene Expression in the Pacific Oyster Crassostrea gigas using Long Double-stranded RNA-mediated RNA Interference

RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectively downregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

Molecular characterization and expression patterns of the big defensin gene in freshwater mussel (Hyriopsis cumingii)

Antimicrobial peptides (AMPs), of which big defensins are examples, are an important component of the natural defenses of most living organisms, and possess remarkable microbicidal activities. In the present study, using expressed-sequence tag sequences from a cDNA library and RACE, the full-length cDNA sequence of the big defensin gene from the triangle-shell pearl mussel, Hyriopsis cumingii, (HcBD), was cloned. The gene consists of a 5'-untranslated region (UTR) of 166 bp, a 3'-UTR region of 96 bp, and an open reading frame of 342 bp that encodes 113 amino acids, consisting of a 23 amino acid signal peptide and a mature peptide of 90 amino acids with a molecular mass of 12.5 kDa. Amino acid sequence analysis showed that the sequence contained a transmembrane domain and a hydrophobic region. The full-length amino acid sequence showed the highest similarity to an amphioxus (Branchiostoma floridae) sequence (64%), and lower similarities to other known defensins (α-, β-, and θ-defensins, and insect defensins). Expression of HcBD was relatively high in the mantle and blood, lower in other tested tissues, and absent in gill and foot tissues. Real-time quantitative PCR was used to investigate HcBD expression in various tissues at different time points after injection of Aeromonas hydrophila. At 4 h post-inoculation, HcBD expression in the mantle, liver, intestine, gill, and foot was greater than in the control, with the greatest expression at 72 h, while at 24 h, expression in the liver, intestine, gill, and foot were at their lowest levels. These results suggest that HcBD might play an important role in the host immune response. This study enriches the basic research on the big defensin family of antimicrobial peptides and lays foundations for further research on antimicrobial peptide expression and relevance to disease defense.

Antimicrobial Activity of Defensins and Defensin-Like Peptides with Special Emphasis on those from Fungi and Invertebrate Animals

Living organisms are in perpetual contact with pathogenic microbes, and in encounter with parasites and predators. In order to protect themselves, they produce a variety of antimicrobial proteins and peptides. One family of such protective or defensive proteins is known as defensins, characterized by a cationic character, a low molecular mass, and an abundance of cysteine residues. Defensins from mammals and plants have been succinctly reviewed by a number of experts in this ever-growing field. This review encompasses the defensin plectasin from the saprophytic fungus Pseudoplectania nigrella as well as defensins and defensin-like peptides from invertebrate animals such as jellyfish, sponges, nematodes, crustaceans, arachnids, insects, bivalves, snails, and sea urchins. Big defensins from mollusks are mentioned together with amphioxus big defensin. The structures and activities of these defense proteins are discussed.

Characterisation of immune-related gene expression in clam (Venerupis philippinarum) under exposure to di(2-ethylhexyl) phthalate

Di(2-ethylhexyl) phthalate (DEHP) mediates the immune system mainly by triggering the production of reactive oxygen species (ROS) and nitric oxide (NO) in higher animals. In the present study, spatial variation in the expression of immune-related genes in clam (Venerupis philippinarum) under acute short-term DEHP treatment was assessed by qPCR. The expression of six genes including glutamine synthetase (GS), IkB (IK), transcription factor activator protein-1 (AP-1), cyclophilin A-1 (CypA-1), heat shock protein 90 (HSP90) and superoxide dismutase (SOD) was dose-dependent. A negative correlation between expression and DEHP treatment was observed for big defensin (BD), glutathione S-transferase (GST), and thioredoxin peroxidase (TP). Surprisingly, lysozyme (LYZ) exhibited two distinct expression patterns at two DEHP doses. Significant differences between the experimental and control groups were observed for all tested genes at the various time points. Overall, our results revealed that DEHP mediates immune responses in clams by various means, and certain genes are promising candidate for biomarkers in DEHP monitoring.

Molecular characterization and expression of a novel big defensin (Sb-BDef1) from ark shell, Scapharca broughtonii

Big defensins, endogenous cysteine-rich antimicrobial peptides (AMPs) with antimicrobial activity and immunomodulatory property, play crucial roles in host defense against various microbial pathogens. A novel big defensin (Sb-BDef1) of ark shell Scapharca broughtonii was identified by expressed sequence tag (EST) and RACE techniques. The Sb-BDef1 cDNA contained an open reading frame (ORF) of 336-bp encoding a polypeptide of 111 amino acids with a putative signal peptide of 21 amino acid residues, followed by a putative propeptide of 11 residues and a putative mature peptide of 79 residues. The mature peptide shared the common features of big defensins, including a high hydrophobic residues region (59%) in the N-terminus, a defensin domain in the C-terminus, which perfectly corresponds to the six conserved disulfide-bonded cysteine residues involved in the formation of the internal disulfide bridges (C1–C5, C2–C4 and C3–C6) in all big defensins from mollusk, horseshoe crab and amphioxus. Quantitative real-time PCR analysis revealed that the expression of Sb-BDef1 transcript was detected in all the tissues examined from normal ark shells, and the temporal expression of Sb-BDef1 mRNA was remarkably up-regulated at 8, 16 h in hemocytes, and at 16, 24 h in hepatopancreas after Vibrio anguillarum-challenge, respectively. These results suggested that Sb-BDef1 was a constitutive and inducible acute-phase protein and should be involved in immune response of Gram-negative microbial infection in ark shell S. broughtonii.

The first chordate big defensin: Identification, expression and bioactivity

Defensins are broadly present in plants, invertebrates and vertebrates, but little information is available about it in amphioxus, a protochordate holding a key phylogenetic position. In this study, a big defensin cDNA was identified from the amphioxus Branchiostoma japonicum (termed Bjbd). The cDNA contained an open reading frame (ORF) of 354 bp encoding a 117 amino acid protein, which had an N-terminal signal sequence followed by a propeptide and the mature big defensin. The mature peptide had the hydrophobic region GAAAVT(A)AA at N-terminus and the consensus pattern C-X6-C-X3-C-X13(14)-C-X4-C-C at C-terminus as well as four α-helices, four β-sheets, and three disulfide bridges (C1–C5, C2–C4 and C3–C6) in the predicted tertiary structure. This is the first big defensin gene ever identified in the phylum Chordata. Quantitative real-time PCR analysis revealed that Bjbd was constitutively expressed in most of the tissues examined, and its expression was remarkably up-regulated following the challenge with LPS, LTA, Aeromonas hydrophila and Staphylococcus aureus. Moreover, the recombinant BjBD was shown to be able to inhibit the growth of S. aureus, Escherichia coli and A. hydrophila. Taken together, these suggest that BjBD is a molecule involved in the removal of invading pathogens.

The Antimicrobial Defense of the Pacific Oyster, Crassostrea gigas. How Diversity may Compensate for Scarcity in the Regulation of Resident/Pathogenic Microflora

Healthy oysters are inhabited by abundant microbial communities that vary with environmental conditions and coexist with immunocompetent cells in the circulatory system. In Crassostrea gigas oysters, the antimicrobial response, which is believed to control pathogens and commensals, relies on potent oxygen-dependent reactions and on antimicrobial peptides/proteins (AMPs) produced at low concentrations by epithelial cells and/or circulating hemocytes. In non-diseased oysters, hemocytes express basal levels of defensins (Cg-Defs) and proline-rich peptides (Cg-Prps). When the bacterial load dramatically increases in oyster tissues, both AMP families are driven to sites of infection by major hemocyte movements, together with bactericidal permeability/increasing proteins (Cg-BPIs) and given forms of big defensins (Cg-BigDef), whose expression in hemocytes is induced by infection. Co-localization of AMPs at sites of infection could be determinant in limiting invasion as synergies take place between peptide families, a phenomenon which is potentiated by the considerable diversity of AMP sequences. Besides, diversity occurs at the level of oyster AMP mechanisms of action, which range from membrane lysis for Cg-BPI to inhibition of metabolic pathways for Cg-Defs. The combination of such different mechanisms of action may account for the synergistic activities observed and compensate for the low peptide concentrations in C. gigas cells and tissues. To overcome the oyster antimicrobial response, oyster pathogens have developed subtle mechanisms of resistance and evasion. Thus, some Vibrio strains pathogenic for oysters are equipped with AMP-sensing systems that trigger resistance. More generally, the known oyster pathogenic vibrios have evolved strategies to evade intracellular killing through phagocytosis and the associated oxidative burst.

α‐Defensins in human innate immunity

Defensins are small, multifunctional cationic peptides. They typically contain six conserved cysteines whose three intramolecular disulfides stabilize a largely β-sheet structure. This review of human α-defensins begins by describing their evolution, including their likely relationship to the Big Defensins of invertebrates, and their kinship to the β-defensin peptides of many if not all vertebrates, and the θ-defensins found in certain non-human primates. We provide a short history of the search for leukocyte-derived microbicidal molecules, emphasizing the roles played by luck (good), preconceived notions (mostly bad), and proper timing (essential). The antimicrobial, antiviral, antitoxic, and binding properties of human α-defensins are summarized. The structural features of α-defensins are described extensively and their functional contributions are assessed. The properties of HD6, an enigmatic Paneth cell α-defensin, are contrasted with those of the four myeloid α-defensins (HNP1-4) and of HD5, the other α-defensin of human Paneth cells. The review ends with a decalogue that may assist researchers or students interested in α-defensins and related aspects of neutrophil function.

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

BackgroundBig defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections.FindingsUsing the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid.ConclusionsWe provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression.

Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis

Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity of invertebrates, preventing the invasion of potential pathogens. Mussels can express a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes. Based on deep RNA sequencing of Mytilus galloprovincialis, we describe the identification, molecular diversity and constitutive expression in different tissues of five novel transcripts pertaining to the macin family (named mytimacins) and eight novel transcripts pertaining to the big defensins family (named MgBDs). The predicted antimicrobial peptides exhibit a N-terminal signal peptide, a positive net charge and a high content in cysteines, allegedly organized in intra-molecular disulfide bridges. Mytimacins and big defensins therefore represent two novel AMP families of M. galloprovincialis which extend the repertoire of cysteine-rich AMPs in this bivalve mollusk.

Molecular Characterization of a Novel Big Defensin from Clam Venerupis philippinarum

Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.