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Bactericidal permeability-increasing protein (BPI) is an antimicrobial protein with potent endotoxin-neutralising activity and plays a crucial role in innate immunity against bacterial infection. In the present study, a bpi (designed as rpbpi) was identified and characterized from manila clam Ruditapes philippinarum. Multiple alignments and phylogenetic analysis suggested that rpbpi was a new member of the bpis family. In non-stimulated clams, rpbpi transcripts were ubiquitously expressed in all tested tissues with the highest expression level in hemocytes. After Vibrio anguillarum challenge, the expression levels of rpbpi mRNA in hemocytes were up-regulated significantly at 3 h and 48 h compared with that in the control, which were 4.01- and 19.10-fold (P < 0.05), respectively. The recombinant RpBPI (rRpBPI) showed high antibacterial activities against Gram-negative bacteria Escherichia coli and V. anguillarum , but not Staphylococcus aureus. Moreover, membrane integrity analysis revealed that rRpBPI increased the membrane permeability of Gram-negative bacteria, and then resulted in cell death. Overall, our results suggested that RpBPI played an important role in the elimination of invaded bacteria through membrane-disruptive activity. • A bpi gene was identified from the manila clam Ruditapes philippinarum. • The expression of rpbpi transcripts was enhanced after Vibrio anguillarum stimulation in vivo. • RpBPI showed high antibacterial activities against Gram-negative bacteria. • RpBPI could modulate the defensive activities of manila clam. [ABSTRACT FROM AUTHOR]

In the present study, a macin was cloned and characterized from clam Venerupis philippinarum (designed as VpMacin). The full-length cDNA of VpMacin was of 579 bp, encoding a peptide of 87 amino acids with the predicted molecular weight of 9.7 kDa. Analysis of the conserved domain suggested that VpMacin was a new member of the macin family. In non-stimulated clams, VpMacin transcripts exhibited different tissue expression pattern, and highly expressed in the tissues of gills and hepatopancreas. Generally, the temporal expression of VpMacin transcripts was significantly induced in hemocytes of clams post Vibrio anguillarum challenge. Moreover, the recombinant VpMacin protein (rVpMacin) showed obvious antimicrobial activities against Gram-positive and Gram-negative bacteria. After incubated with 40 μM rVpMacin, all detected Escherichia coli could be killed within 60 min. Membrane integrity analysis revealed that rVpMacin could increase the membrane permeability of bacteria and then resulted in cell death. Overall, our results suggested that VpMacin had an important function in host defense against invasive pathogens. Image 1 • A macin was identified from the manila clam Venerupis philippinarum. • VpMacin showed obvious antimicrobial activities against both Gram-positive and Gram-negative bacteria. • VpMacin could eliminate invading bacteria through membrane-disruptive activities. [ABSTRACT FROM AUTHOR]

Abstract Peptidoglycan recognition proteins (PGRPs) are important pattern recognition receptors in the innate immune system of invertebrates. In the study, a short PGRP (designed as RpPGRP) was identified and characterized from the manila clam Ruditapes philippinarum. The open reading frame of RpPGRP encoded a polypeptide of 249-amino acids with a calculated molecular mass of 27.2 kDa and an isoelectric point of 6.62. Multiple alignments and phylogenetic analysis strongly suggested that RpPGRP was a new member of the PGRP superfamily. In non-stimulated clams, RpPGRP exhibited different tissue expression pattern, and highly expressed in hepatopancreas and hemocytes. Expression of RpPGRP transcripts was significantly up-regulated in hemocytes of clams post Vibrio anguillarum or Micrococcus luteus challenge. The recombinant RpPGRP (rRpPGRP) exhibited high affinity to PGN, LPS and zymosan in a concentration-dependent manner. With a broad spectrum of bacterial binding activities, rRpPGRP exhibited strong agglutination activity to Escherichia coli , Vibrio splendidus , V. anguillarum and M. luteus. Furthermore, rRpPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble PGN. Especially, rRpPGRP exhibited significant antibacterial activity against E. coli and M. luteus. Moreover, the biofilm formation of E. coli could be inhibited after rRpPGRP incubation in the presence of Zn2+. This inhibitory effect of rRpPGRP might attribute to its amide bactericidal activity. Taken together, rRpPGRP played important roles in PGRP-mediated immune defense mechanisms, especially by recognizing antigens and eliminating bacteria. Highlights • A short peptidoglycan recognition protein (PGRP) was identified from the manila clam Ruditapes philippinarum. • RpPGRP transcripts were highly expressed in hepatopancreas and hemocytes of healthy manila clam. • RpPGRP functioned as a bactericidal amidase to degrade PGN and inhibited the growth of tested bacteria. [ABSTRACT FROM AUTHOR]

Abstract In the present study, a scavenger receptor class B type I (designed as RpSR-BI) was cloned and characterized from manila clam Ruditapes philippinarum. The full-length cDNA of RpSR-BI was of 2000 bp, containing an open reading frame (ORF) of 1515 bp. Multiple alignments and phylogenetic analysis strongly suggested that RpSR-BI was a member of the scavenger receptors family. The mRNA transcript of RpSR-BI was constitutively expressed in all tested tissues, and mainly expressed in hepatopancreas and hemocytes. Generally, Vibrio anguillarum or Micrococcus luteus challenge induced the expression of RpSR-BI transcripts in hemocytes of manila clams. Recombinant protein of RpSR-BI (rRpSR-BI) could bind lipopolysaccharides, peptidoglycan and glucan, but not chitin in vitro. Coinciding with the PAMPs binding assay, a broad agglutination spectrum was displayed by rRpSR-BI including Gram-positive bacteria and Gram-negative bacteria. Moreover, rRpSR-BI could enhance the phagocytosis and chemotaxis of hemocytes. These results showed that RpSR-BI functioned as a pattern recognition receptor (PRR) with distinct recognition spectrum, and also as an opsonin involved in the innate immune response of R. philippinarum. Highlights • A scavenger receptor class B, type I (SR-BI) was identified from the manila clam Ruditapes philippinarum. • The expression of RpSR-BI transcripts was up-regulated post V. anguillarum and M. luteus challenge. • RpSR-BI probably served as an opsonin in the innate immunity of manila clam. [ABSTRACT FROM AUTHOR]

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum. • A defensin isoform was identified from manila clam Ruditapes philippinarum. • RRpdef1α showed broad antimicrobial activities against Gram-positive and Gram-negative bacteria. • RRpdef1α was capable of causing bacterial membrane permeabilization. • RRpdef1α could function as an opsonin to promote phagocytosis and chemotaxis of hemocytes in vitro. [ABSTRACT FROM AUTHOR]

Abstract Fas-associated protein with death domain (FADD) is an essential element in cell death, and also implicates in cell cycle progression, inflammation and innate immunity. In the study, an FADD (designated as RpFADD) was identified and characterized from manila clam, Ruditapes philippinarum. Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family. The RpFADD transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After challenged with Vibrio anguillarum or Micrococcus luteus , the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72 h and 48 h, respectively. Knockdown of RpFADD down-regulated the transcript levels of RpIKK, RpTAK1 and RpNF-κB with the exception of RpIκB. Moreover, RpFADD primarily localized in the cell cytoplasm, and its over-expression promoted the apoptosis of HeLa cells. These results revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFADD in innate immunity. Highlights • A Fas-associated protein with death domain (FADD) was identified from the manila clam Ruditapes philippinarum. • RpFADD was differentially up-regulated when challenged with Vibrio anguillarum or Micrococcus luteus. • RpFADD primarily localized in the cell cytoplasm and its over-expression promoted the apoptosis of HeLa cells. [ABSTRACT FROM AUTHOR]

In the present study, a histone H2A (designed as RpH2A) was identified and characterized from clam Ruditapes philippinarum , and its open reading frame (ORF) was of 387 bp encoding a polypeptide of 128 amino acids. The deduced amino acid sequence of RpH2A shared high identities ranging from 57.1% to 96.1% with that of other identified H2A. The mRNA expression of RpH2A was up-regulated significantly after Vibrio anguillarum challenge. The recombinant RpH2A protein (rRpH2A) displayed significantly binding affinity to lipopolysaccharide (LPS) and peptidoglycan (PGN) in vitro , and also exhibited antimicrobial properties against Escherichia coli. In addition, the antimicrobial RpH2A was shown to co-localize with extracellular traps (ETs) released from hemocytes induced by E. coli , suggesting that RpH2A might contribute to eliminate invading bacteria in clam ETs. Altogether, our data revealed that RpH2A could function as antimicrobial peptides, which might play a crucial role in the immune responses of hemocytes ETs in clams. • A conserved H2A histone (RpH2A) was identified from Ruditapes philippinarum. • rRpH2A displayed significantly binding activity to both LPS and PGN. • rRpH2A exhibited antimicrobial properties against Escherichia coli. • RpH2A was a component of extracellular traps (ETs) released from hemocytes after E. coli infection. [ABSTRACT FROM AUTHOR]

[Display omitted] • macin was identified from the abalone Haliotis discus hannai. • HdMac was highly expressed in hemocytes and hepatopancreas. • HdMac exhibited antibacterial activities against Gram-negative bacteria, especially for Vibrio anguillarum. • HdMac eliminated invading bacteria through membrane-disruptive activities. Macins are a family of antimicrobial peptides, which play multiple roles in the elimination of invading pathogens. In the present study, a macin was cloned and characterized from Pacific abalone Haliotis discus hannai (Designated as HdMac). Analysis of the conserved domain suggested that HdMac was a new member of the macin family. In non-stimulated abalones, HdMac transcripts were constitutively expressed in all five tested tissues, especially in hemocytes. After Vibrio harveyi stimulation, the expression of HdMac mRNA in hemocytes was significantly up-regulated at 12 hr (P < 0.01). RNAi-mediated knockdown of HdMac transcripts affected the survival rates of abalone against V. harveyi. Moreover, recombinant protein of HdMac (rHdMac) exhibited high antibacterial activities against invading bacteria, especially for Vibrio anguillarum. In addition, rHdMac possessed binding activities towards glucan, lipopolysaccharides (LPS), and peptidoglycan (PGN), but not chitin in vitro. Membrane integrity analysis revealed that rHdMac could increase the membrane permeability of bacteria. Meanwhile, both the phagocytosis and chemotaxis ability of hemocytes could be significantly enhanced by rHdMac. Overall, the results showed that HdMac could function as a versatile molecule involved in immune responses of H. discus hannai. [ABSTRACT FROM AUTHOR]

In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens. • Two macins were identified from clam Venerupis philippinarum. • Knockdown of VpMacins led to higher cumulative mortalities. • rVpMacin-2 exhibited higher broad-spectrum antimicrobial activities. • rVpMacin-1, but not rVpMacin-2, induced phagocytosis and chemotaxis significantly. [ABSTRACT FROM AUTHOR]

[Display omitted] • A ubiquitin was identified from the manila clam Ruditapes philippinarum. • Recombinant RpUbi showed broad antibacterial activities against Gram-positive and Gram-negative bacteria. • rRpUbi might perform antibacterial activities through DNA-binding process. In the present study, a ubiquitin (designated as RpUbi) was identified and characterized from clam Ruditapes philippinarum. Phylogenetic analysis strongly suggested that RpUbi was a member of the ubiquitin family. In non-stimulated clams, RpUbi transcripts were constitutively expressed in all examined tissues, especially in the gills and hemocytes. After Vibrio anguillarum challenge, expression of RpUbi mRNA in hemocytes was significantly up-regulated. Recombinant RpUbi (rRpUbi) showed high antibacterial activity against Gram-positive and Gram-negative bacteria. Notably, membrane integrity and electrochemical assay indicated that rRpUbi could invade the inner layer. Moreover, DNA migration could be inhibited by rRpUbi in a concentration-dependent manner. In general, our results suggested that RpUbi played an important role in host defense against invading bacteria, perhaps through a DNA-binding process. [ABSTRACT FROM AUTHOR]

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro , whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi , Vibrio splendidus and V. anguillarum , while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum. • Two C-type lectins were identified from the manila clam Ruditapes philippinarum. • VpClecs are C-type lectins with different PAMPs binding spectrum. • VpClecs can agglutinate Gram-positive and Gram-negative bacteria. • VpClecs can function as opsonins to prompt phagocytosis against non-self. [ABSTRACT FROM AUTHOR]

In the study, two c-type lectins were identified and characterized from the manila clam Venerupis philippinarum (designed as VpClec-1 and VpClec-2, respectively). Multiple alignments and phylogenetic analysis strongly suggested that they were new members of the c-type lectin superfamily. In normal tissue of clams, both VpClec-1 and VpClec-2 transcripts were highly expressed in the tissue of hepatopancreas. After Vibrio anguillarum challenge, the temporal expression of both VpClec-1 and VpClec-2 transcripts was up-regulated in the hemocytes of manila clams. The recombinant protein VpClec-1 (rVpClec-1) showed obvious binding activities to lipopolysaccharide (LPS), peptidoglycan (PGN), glucan and zymosan in vitro , while the recombinant protein VpClec-2 (rVpClec-2) could only bind LPS, glucan and zymosan. Coinciding with the PAMPs binding assay, both rVpClec-1 and rVpClec-2 displayed broad agglutination and antibacterial activities towards Vibrio harveyi , Vibrio splendidus , Vibrio anguillarum , Enterobacter cloacae and Aeromonas hydrophila. Moreover, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rVpClec-1 and rVpClec-2. Notably, the rVpClec-1 but not rVpClec-2 elicited a chemotactic response from hemocytes. All the results showed that VpClec-1 and VpClec-2 functioned as pattern recognition receptors (PRRs) with distinct recognition spectrum, and involved in the innate immune responses of manila clams. • Two c-type lectins (VpClec-1 and VpClec-2) were identified from the Manila clam Ruditapes philippinarum. • Both VpClec-1 and VpClec-2 were highly expressed in hepatopancreas of healthy Manila clam. • Both VpClec-1 and VpClec-2 showed a broad spectrum of microbial binding and opsonic activities. [ABSTRACT FROM AUTHOR]

In the present study, a sialic acid-binding lectin was cloned and characterized from Manila clam Ruditapes philippinarum (designed as RpSabl). The open reading frame of RpSabl encoded a polypeptide of 162 amino acids with a calculated molecular mass of 17.7 kDa. Analysis of the conserved domain suggested that RpSabl was a new member of the sialic acid-binding lectins family. In non-stimulated clams, RpSabl transcripts were constitutively expressed in all five tested tissues, especially in hepatopancreas. After Vibrio anguillarum challenge, the expression of RpSabl mRNA in hepatopancreas was significantly up-regulated at 3 h (3.8-fold, P < 0.05), 6 h (4.9-fold, P < 0.05), 12 h (12.3-fold, P < 0.01) and 24 h (9.7-fold, P < 0.01), while RpSabl transcripts in hemocytes was only significantly up-regulated at 6 h (8.5-Fold, P < 0.01). RNAi-mediated knockdown of RpSabl transcripts affected the survival rates of Manila clam against V. anguillarum , perhaps mainly due to the inhibited expression of antibacterial effectors (e.g. lysozyme and defensin). Moreover, recombinant protein of RpSabl (rRpSabl) possessed binding activities towards lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan in vitro. Coinciding with the Pathogen-associated molecular patterns (PAMPs) binding assay, rRpSabl displayed broad bacterial-agglutination properties towards Vibrio harveyi , Vibrio splendidus , V. anguillarum , Enterobacter cloacae and Aeromonas hydrophila. Meanwhile, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rRpSabl incubation. All these results showed that RpSabl could function as a versatile molecule involved in the innate immune responses of R. philippinarum. • A sialic acid binding lectin (Sabl) was identified from the manila clam Ruditapes philippinarum. • Recombinant RpSabl possessed broad bacterial-agglutination properties perhaps benefiting from its PAMPs binding abilities. • RpSabl could function as an opsonin to promote phagocytosis and encapsulation of hemocytes against non-self. [ABSTRACT FROM AUTHOR]

In the present study, a sialic acid-binding lectin was identified and characterized from Manila clam Ruditapes philippinarum (designed as RpSABL-1). Multiple alignments strongly suggested that RpSABL-1 was a new member of the sialic acid-binding lectin family. In non-stimulated clams, RpSABL-1 transcripts were constitutively expressed in all five tested tissues, especially in hepatopancreas. After Vibrio anguillarum challenge, the expression of RpSABL-1 mRNA was significantly up-regulated at 6 h (P < 0.05), 12 h (P < 0.01) and 24 h (P < 0.01). Recombinant RpSABL-1 protein (rRpSABL-1) displayed apparent binding activities towards lipopolysaccharides (LPS) and peptidoglycan (PGN), but not to glucan or chitin in vitro. Coinciding with the PAMPs binding assay, rRpSABL-1 exhibited obvious agglutination activities against Gram-positive bacterium Staphyloccocus aureus , Gram-negative bacteria Escherichia coli, V. anguillarum and Vibrio harveyi. Meanwhile, rRpSABL-1 showed antibacterial activities against E. coli , and biofilm formation of E. coli could also be inhibited after incubated with rRpSABL-1. Moreover, the encapsulation, phagocytosis and chemotactic ability of hemocytes could be enhanced by rRpSABL-1. All these results suggested that RpSABL-1 could function as a pattern recognition receptor with versatile functions in the innate immune responses of R. philippinarum. • A sialic acid binding lectin (SABL) was identified from the manila clam Ruditapes philippinarum. • Recombinant RpSABL-1 showed a broad agglutination spectrum against Gram-positive and Gram-negative bacteria. • RpSABL-1 could function as a pattern recognition receptor with broad recognition spectrum. [ABSTRACT FROM AUTHOR]

Mytimacin-4 was highly expressed in posterior adductor muscle of mussel Mytilus galloprovincialis and was inducible upon Vibrio challenge. Mytimacin-4 protein showed antimicrobial activities against gram-positive and gram-negative bacteria. • Mytimacin-4 was highly expressed in posterior adductor muscle of mussel. • Mytimacin-4 expression was inducible upon Vibrio challenge. • Recombinant mytimacin-4 protein had potent antimicrobial activities. Antimicrobial peptides (AMPs) play fundamental roles in the innate immunity of invertebrates. Mytimacin-4 is a kind of AMP gene previously sequenced from Mytilus galloprovincialis based on an identified EST sequence in our lab. In the present study, the tissue distribution and antimicrobial activities of mytimacin-4 were further investigated. A qRT-PCR analysis revealed that mytimacin-4 transcripts were constitutively expressed in all of the tested tissues of M. galloprovincialis , with the highest expression level in the posterior adductor muscle. After challenge by Vibrio anguillarum , the expression level of mytimacin-4 gene was significantly increased at 24 h (P < 0.05) in the mantle and increased at 48 h (P < 0.05) in the posterior adductor muscle. This finding suggested that mytimacin-4 transcripts were inducible upon pathogen infection. A minimal inhibitory concentration (MIC) assay indicated that recombinant mytimacin-4 protein had potent antimicrobial activities against gram-positive and gram-negative bacteria. Among the tested microorganisms, mytimacin-4 protein exhibited strong inhibition activities against Bacillus subtilis and Vibrio parahaemolyticus with MICs of 0.315 μM and 0.62 μM, respectively. This study provides for the first time direct evidence of antimicrobial action of mytimacin-4 in M. galloprovincialis. [ABSTRACT FROM AUTHOR]

The formation of extracellular traps (ETs) is an important innate immune mechanism that serves to combat different invading pathogens. In this study, zymosan significantly induced the formation of ETs in the hemocytes of Ruditapes philippinarum , and this effect was accompanied by translocation of the mitochondria to the cell surface. Zymosan stimulation clearly induced an increase in intracellular ROS and MPO production and an overexpression of ROS-related genes (PI3K , AKT and HIF). In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Conversely, mitochondrial superoxide inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. In addition, we found that zymosan-induced ETs showed antibacterial activities against gram-negative and gram-positive bacteria, such as Vibrio anguillarum , Vibrio harveyi , Escherichia coli and Micrococcus luteus. Taken together, these findings elucidated a new antibacterial approach for R. philippinarum and highlighted the role of mitochondria in the formation of zymosan-induced ETs. • Zymosan induced extracellular trap (ET) formation in hemocytes of Ruditapes philippinarum. • Zymosan increased the production of ROS and MPO in ETosis. • Mitochondrial ROS are essential in the formation of zymosan-triggered ETs. • Zymosan-induced ETs exhibited antibacterial activities against both gram-negative and gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Extracellular traps (ETs) have been found to be an important strategy of mammals to immobilize and kill invading microorganisms. In the present study, we observed the formation of ETs in the hemocytes of marine mollusks Ruditapes philippinarum in response to challenge from bacteria Vibrio anguillarum , and examined the potential factors and signaling pathways underling this process. We detected an increase of reactive oxygen species (ROS) and myeloperoxidase (MPO) production during ETosis, accompanied by significantly up-regulated expression of ROS-related and MPO genes. The suppression of ETs structures by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium chloride, DPI) and MPO inhibitor (aminobenzoic acid hydrazide, ABAH) further confirmed the essential roles ROS and MPO played in ETosis. Furthermore, ET production could be inhibited by phosphotidylinsitol-3-kinase (PI3K) inhibitor (LY294002) and extracellular regulated protein kinase (ERK) inhibitor (U0126), suggesting the idea that both the PI3K and ERK pathways were suggested to function during ETosis. In addition, the ETosis process was accompanied by enhancement of glycolysis-related enzymatic activities, e.g., pyruvate kinase (PK) and hexokinase (HK), and over-expression of the glycolysis-related genes, e.g., PK, HK and glucose transport protein (GLUT), indicating high involvement of glycolysis in the ETosis process. Furthermore, our scanning electron microscopy (SEM) observation and antibacterial activities test successfully showed the patterns how clam ETs entrapped and killed the invading V. anguillarum. Taken together, our results revealed that ETosis with bactericidal effect increased ROS, MPO and glycolysis level and carried out in a ROS-, MPO-, PI3K-ERK-dependent manner. • Vibrio anguillarum induced extracellular trap (ET) formation in clam hemocytes. • The production of reactive oxygen species (ROS) and myeloperoxidase (MPO) was significantly increased in clam ETosis. • Glycolysis was involved during the production of V. anguillarum -induced ETs. • ETosis was carried out in a ROS-, MPO-, PI3K-ERK-dependent manner. [ABSTRACT FROM AUTHOR]