A novel C1qDC (PoC1qDC) with a collagen domain in Paralichthys olivaceus mediates complement activation and against bacterial infection.

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection.

Screening of intestinal probiotics and the effects of feeding probiotics on the growth, immune, digestive enzyme activity and intestinal flora of Litopenaeus vannamei.

In this experiment, 426 strains were isolated from the intestinal tract of Litopenaeus vannamei, and 11 strains showed strong digestive enzyme production activity and antagonistic effect against common bacterial pathogens of shrimp. After hemolysis activity test and drug sensitivity test, 2 candidate probiotics with good bacteriostatic activity, strong enzyme production ability and relatively sensitive to antibiotics were screened out, and were identified by 16s rDNA molecular identification and Biolog-System as Enterobacter hominis (E3) and lactobacillus (L3). First, the biological characteristics of 2 candidate probiotics were studied. The optimum growth conditions of E3: temperature, 30 °C; pH, 8.0; NaCl, 2.5%; bovine bile salt, 0.15%; and the optimum growth conditions of L3: temperature, 40 °C; pH, 6.0; NaCl, 0.5%; bovine bile salt, 0.0015%. Secondly, a 28-day feeding experiment was conducted using probiotic concentration of 107 CFU g-1 to determine the changes of the activities of blood related immune enzymes (SOD, PPO, ACP, POD, CAT, LZM) and intestinal digestive enzymes (NP, AL, LPS) during the feeding process of shrimp, the results showed that during the course of feeding, the activities of immune enzyme and digestive enzyme of shrimp fed with probiotics showed an increasing trend, and the growth rate of body weight of shrimp was higher than that of control group. After feeding, the cumulative mortality of probiotics groups were significantly lower than that of the control group after WSSV infection. And the mid-gut of L. vannamei was observed by electron microscope, the results showed that the intestinal mucosa was tight and the epithelium cells showed an active secretory state in probiotics group. Finally, the intestinal microbial communities of shrimp were compared and analyzed by using Biolog-ECO method in the later period of feeding, the results showed: compared with the control group, the average color change rate of the experimental group fed with probiotics increased significantly, indicating that probiotics enhanced the intestinal microorganism activity; The ability of intestinal microorganism to utilize carbon source was significantly enhanced in the experimental group, which indicated that the digestive enzyme secreted by probiotics could improve the digestion and absorption rate of prawn feed, thus promoting the rapid growth of shrimp; The Shannon index, Simpson index and McIntosh index of probiotics groups showed significant difference in 1st and 5th days, but tended to be the same in the 10th day, the results showed that probiotics could maintain in L. vannamei intestines at least 5 days.

Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis.

Glycogen plays an important role in glucose and energy homeostasis at cellular and organismal levels. In glycogen synthesis, glycogen synthase (GS) is a rate-limiting enzyme catalysing the addition of α-1,4-linked glucose units from (UDP)3-glucose to a nascent glycogen chain using glycogenin (GN) as a primer. While studies on mammalian liver GS (GYS2) are numerous, enzymes from crustaceans, which also use glycogen and glucose as their main energy source, have received less attention. In the present study, we amplified full-length GS cDNA from Eriocheir sinensis. Tissue expression profiling revealed the highest expression of GS in the hepatopancreas. During moulting, GS expression and activity declined, and glycogen levels in the hepatopancreas were reduced. Recombinant GS was expressed in Escherichia coli Rosetta (DE3), and induction at 37°C or 16°C yielded EsGS in insoluble inclusion bodies (EsGS-I) or in soluble form (EsGS-S), respectively. Enzyme activity was measured in a cell-free system containing glucose-6-phosphate (G6P), and both forms possessed glycosyltransferase activity, but refolded EsGS-I was more active. Enzyme activity of both GS and EsGS-I in the hepatopancreas was optimum at 25°C, which is coincident with the optimum growth temperature of Chinese mitten crab, and higher (37°C) or lower (16°C) temperatures resulted in lower enzyme activity. Taken together, the results suggest that GS may be important for maintaining normal physiological functions such as growth and reproduction.

EsGLUT4 and CHHBP are involved in the regulation of glucose homeostasis in the crustacean Eriocheir sinensis.

Glucose is an essential energy source for both vertebrates and invertebrates. In mammals, glucose uptake is mediated primarily by glucose transporters (GLUTs), members of the major facilitator superfamily (MFS) of passive transporters. Among the GLUTs, GLUT4 is the main glucose transporter in muscles and adipocytes. In skeletal muscle cells, GLUT4 interacts with the lipid raft protein flotillin to transport glucose upon stimulation by insulin. Although several studies have examined GLUT4 function in mammals, few have been performed in crustaceans, which also use glucose as their main energy source. Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormone found only in arthropods, and one of its roles is to regulate glucose homeostasis. However, the molecular mechanism that underlies CHH regulation and whether GLUT4 is involved in its regulation in crustaceans remain unclear. In the present study, we identified a full-length GLUT4 cDNA sequence (defined herein as EsGLUT4) from the Chinese mitten crab Eriocheir sinensis and analyzed its tissue distribution and cellular localization. By the ForteBio Octet system, two large hydrophilic regions within EsGLUT4 were found to interact with the CHH binding protein (CHHBP), an E. sinensis flotillin-like protein. Interestingly, live-cell imaging indicated that EsGLUT4 and CHHBP responded simultaneously upon stimulation by CHH, resulting in glucose release. In contrast to insulin-dependent GLUT4, however, EsGLUT4 and CHHBP were present within cytoplasmic vesicles, both translocating to the plasma membrane upon CHH stimulation. In conclusion, our results provide new evidence for the involvement of EsGLUT4 and CHHBP in the regulation of glucose homeostasis in crustacean carbohydrate metabolism.

CHHBP: a newly identified receptor of crustacean hyperglycemic hormone.

Crustacean hyperglycemic hormone (CHH) is a neurohormone found only in arthropods that plays a pivotal role in the regulation of hemolymph glucose levels, molting and stress responses. Although it was determined that a membrane guanylyl cyclase (GC) acts as the CHH receptor in the Y-organ during ecdysteroidogenesis, the identity of the CHH receptor in the hepatopancreas has not been established. In this study, we identified CHH binding protein (CHHBP), as a potential receptor by screening the annotated unigenes from the transcriptome of ITALIC! Eriocheir sinensis, after removal of the eyestalk. Analysis of the binding affinity between CHH and CHHBP provided direct evidence that CHH interacts with CHHBP in a specific binding mode. Subsequent analysis showed that CHHBP is expressed primarily in the hepatopancreas where it localizes to the cell membrane. In addition, real-time PCR analysis showed that ITALIC! CHHBPtranscript levels gradually increase in the hepatopancreas following eyestalk ablation. RNAi-mediated suppression of ITALIC! CHHBPexpression resulted in decreased glucose levels. Furthermore, the reduction of blood glucose induced by ITALIC! CHHBPRNAi reached the same level as that observed in the eyestalk ablation group, suggesting that CHHBP is involved in glucose metabolism regulated by CHH. In addition, compared with the control group, injection of CHH was unable to rescue the decreased glucose levels in ITALIC! CHHBPRNAi crabs. CHH induced transport of 2-NBDG to the outside of cells, with indispensable assistance from CHHBP. Taken together, these findings suggest that CHHBP acts as one type of the primary signal processor of CHH-mediated regulation of cellular glucose metabolism.

Facile synthesis of multifunctional multi-walled carbon nanotube for pathogen Vibrio alginolyticus detection in fishery and environmental samples.

Interest in carbon nanotubes for detecting the presence of pathogens arises because of developments in chemical vapor deposition synthesis and progresses in biomolecular modification. Here we reported the facile synthesis of multi-walled carbon nanotubes (MWCNTs), which functioned as immuno-, magnetic, fluorescent sensors in detecting Vibrio alginolyticus (Va). The structures and properties of functionalized MWCNTs were characterized by ultraviolet (UV), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), magnetic property measurement system (MPMS) and fluorescent spectra (FL). It was found that the functionalized MWCNTs showed: (1) low nonspecific adsorption for antibody-antigen, (2) strong interaction with antibody, and (3) high immune-magnetic activity for pathogenic cells. Further investigations revealed a strong positive linear relationship (R=0.9912) between the fluorescence intensity and the concentration of Va in the range of 9.0 × 10(2) to 1.5 × 10(6) cfum L(-1). Moreover, the relative standard deviation for 11 replicate detections of 1.0 × 10(4) cfum L(-1) Va was 2.4%, and no cross-reaction with the other four strains was found, indicating a good specificity for Va detection. These results demonstrated the remarkable advantages of the multifunctional MWCNTs, which offer great potential for the rapid, sensitive and quantitative detection of Va in fishery and environmental samples.

Molecular characterization and expression analysis of Toll-like receptor 21 cDNA from Paralichthys olivaceus.

Toll-like receptor (TLR) is believed to play crucial role in host defense of pathogenic microbes in innate immune system. In the present study, the full-length cDNA of Paralichthys olivaceus Toll-like receptor 21 (Po-TLR21) was cloned by homology cloning and rapid amplification of cDNA ends (RACE) technique. The Po-TLR21 cDNA sequence was 3687 bp, containing an open reading frame of 2922 bp encoding 973 amino acids. TMHMM and SMART program analysis indicated that protein contained one transmembrane domain, eighteen leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). Multiple alignment analysis of the Po-TLR21 protein-coding sequence with other known TLR21 from grouper, pufferfish, zebrafish, cod, catfish, carp and chicken showed the homology of 67%, 63%, 54%, 52%, 51%, 49%, and 39%, respectively. The Po-TLR21 mRNA expression patterns were measured by real-time PCR. The results revealed that TLR21 is widely expressed in various tested healthy tissues, and highly expressed in spleen and gill. In vivo immunostimulation experiments revealed that expression of TLR21 is modulated by Vibrio anguillarum (V. anguillarum), CpG oligodeoxynucleotides (CpG ODN) and poly I:C. Moreover, the inhibitor of homodimerization of myeloid differentiation factor 88 (MyD88) could significantly reduce the up-regulation of TLR21, MyD88, and tumor necrosis factor (TNF) expression in CpG ODN or poly I:C-treated head kidney cells in vitro. These results indicate that TLR21 may be involved in the pathogen recognition in the early innate immune.

Cynoglossus semilaevis thioredoxin: a reductase and an antioxidant with immunostimulatory property.

Thioredoxin (Trx) is a small redox protein existing ubiquitously in all living organisms and plays an important role in multiple cellular processes, including transcriptional regulation and immune response. To date very few studies have been carried out to examine the function of piscine Trx. In this study, we identified and analyzed the function of a Trx homologue, CsTrx1, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsTrx1 is composed of 107 residues and shares 54.1-60.8% overall identities with the Trx of other teleosts. CsTrx1 contains the highly conserved CXXC motif, which in mammals is known to be the active site, in the form of CQPC. Expression of CsTrx1 as determined by quantitative real-time reverse transcriptase PCR was highest in liver and upregulated in time-dependent manners by bacterial infection and by exposure to iron, copper, and hydrogen peroxide. Purified recombinant CsTrx1 (rCsTrx1) exhibited insulin disulfide reductase activity and antioxidant activity, both which, however, were lost when the two cysteine residues in the CQPC motif were mutated to serine. Further analysis showed that rCsTrx1 was able to stimulate the proliferation of head kidney leukocytes, upregulate the expression of immune relevant genes, and enhance the resistance of leukocytes against bacterial infection. Taken together, these results indicate that CsTrx1 is a biologically active reductase and an antioxidant that requires the CXXC motif for activity and that CsTrx1 possesses cytokine-like immunoregulatory property. These results suggest a role for CsTrx1 in protecting cells against oxidative stress caused by oxidant exposure and pathogen infection.

CsCXCe1: A novel Cynoglossus semilaevis CXC chemokine that functions as a chemoattractant and an immunomodulator for peripheral blood leukocytes.

Chemokines are small cytokines that, based on their structural differences, are classified into four groups, one of which is called CXC chemokines. In this study, we identified a CXC chemokine, CsCXCe1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its function. The deduced amino acid sequence of CsCXCe1 contains 115 residues and is phylogenetically distinct from known CXC chemokines. CsCXCe1 possesses the conserved RCXC motif in the form of RCWC but lacks the ELR sequence that is found in some CXC chemokines. Expression of CsCXCe1 as determined by quantitative real time RT-PCR occurred abundantly in immune organs and was upregulated by bacterial and viral infection in time dependent manners. Purified recombinant CsCXCe1 (rCsCXCe1) exhibited comparable chemotactic activities against tongue sole and turbot (Scophthalmus maximus) peripheral blood leukocytes (PBL). Microscopic analysis identified lymphocytes as the major cellular population in PBL that responds to rCsCXCe1. Mutational study showed that when the two cysteine residues in the RCWC motif of CsCXCe1 were substituted by serine, the chemoattractive activity of CsCXCe1 was completely lost. Further study showed that treatment of PBL with rCsCXCe1 (i) stimulated cellular proliferation and respiratory burst activity, (ii) upregulated the expression of a wide spectrum of immune relevant genes, and (iii) enhanced cellular resistance against bacterial infection. Taken together, these results indicate that CsCXCe1 is likely a new type of CXC chemokine that exerts chemotactic and immunostimulatory effects on PBL.

An inflammatory CC chemokine of Cynoglossus semilaevis is involved in immune defense against bacterial infection.

Chemokines are a family of small cytokines that regulate leukocyte migration. Based on the arrangement of the first two cysteine residues, chemokines are classified into four groups called CXC(α), CC(ß), C, and CX(3)C. In this study, we identified a CC chemokine, CsCCK1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its biological activity. The deduced amino acid sequence of CsCCK1 contains 111 amino acid residues and is phylogenetically belonging to the CCL19/21/25 group of CC chemokines. CsCCK1 possesses a DCCL motif that is highly conserved among CC chemokines. Quantitative real time RT-PCR analysis showed that the expression of CsCCK1 was relatively abundant in immune organs under normal physiological conditions and was upregulated by experimental infection of a bacterial pathogen. Purified recombinant CsCCK1 (rCsCCK1) induced chemotaxis in peripheral blood leukocytes (PBL) of both tongue sole and turbot (Scophthalmus maximus) in a dose-dependent manner. Mutation of the CC residues in the DCCL motif by serine substitution completely abolished the biological activity of rCsCCK1. When rCsCCK1, but not the mutant protein, was added to the cell culture of PBL, it enhanced cellular resistance against intracellular bacterial infection. Taken together, these results indicate that CsCCK1 is a functional CC chemokine whose biological activity depends on the DCCL motif and that CsCCK1 plays a role in host immune defense against bacterial infection.

The high mobility group box 1 protein of Sciaenops ocellatus is a secreted cytokine that stimulates macrophage activation.

High mobility group box 1 protein (HMGB1) is a chromatin-associated nonhistone protein that is involved in nucleosome formation and transcriptional regulation. In addition, HMGB1 is also known as an extracellular cytokine that triggers inflammation and immune responses. HMGB1-like sequences have been identified in a number of fish species, however, the function of piscine HMGB1 remains uninvestigated. In this study, we reported the identification and analysis of SoHMGB1, an HMGB1 homologue from red drum (Sciaenops ocellatus). SoHMGB1 is 206 residues in length and contains two basic HMG boxes and a highly acidic C-terminal domain. SoHMGB1 shares 71-87% overall sequence identities with the HMGB1 counterparts from human, rat, and several fish species. Quantitative real time RT-PCR analysis showed that constitutive SoHMGB1 expression was detected in various tissues, with the lowest and highest levels found in kidney and muscle respectively. Bacterial challenge upregulated SoHMGB1 expression in head kidney (HK) and HK macrophages and induced extracellular secretion of SoHMGB1 by the activated macrophages. Recombinant SoHMGB1 (rSoHMGB1) purified from yeast exhibited no direct antimicrobial effect but was significantly stimulatory on the proliferation, activation, and bactericidal activity of HK macrophages. Taken together, these results indicate for the first time that a fish HMGB1, SoHMGB1, can function as a secreted cytokine in the event of bacterial infection and promote innate defense through the activation of macrophages.

Interleukin-8 of Cynoglossus semilaevis is a chemoattractant with immunoregulatory property.

Interleukin-8 (IL-8), or CXCL8, is a member of the CXC chemokine family that in mammals is known to mediate inflammatory response. In this study, we identified and analyzed an IL-8 orthologue, CsIL8, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsIL8 contains 100 residues and is closely related to the lineage 1 IL-8 of a number of fish species. In silico analysis identified in CsIL8 a CXC chemokine domain that contains four conserved cysteine residues, two of which form the CXC signature motif of CXC chemokines. Expression of CsIL8 as determined by quantitative real time RT-PCR was detected in a wide range of tissues under normal physiological conditions and was upregulated by bacterial challenge and by vaccination with a subunit vaccine. Purified recombinant CsIL8 (rCsIL8) induced migration of peripheral blood leukocytes and head kidney (HK) lymphocytes and stimulated the proliferation of these cells in a dose-dependent manner. When rCsIL8 was added to the cell culture of HK lymphocytes, it upregulated the expression of interleukin-1ß and CsIL8 in a time-dependent fashion. These results indicate that CsIL8 is a biologically active CXC chemokine with immunoregulatory activity and that CsIL8 is involved in pathogen-induced inflammatory response.

The g-type lysozyme of Scophthalmus maximus has a broad substrate spectrum and is involved in the immune response against bacterial infection.

Lysozyme is a muramidase that inflicts damage on bacterial cell wall by catalyzing the cleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan. Lysozymes are classified into several types, one of which is the goose-type (g-type). In this study, we identified and analyzed a g-type lysozyme (SmLysG) from turbot Scophthalmus maximus. The deduced amino acid sequence of SmLysG contains 193 residues and is most closely related to that of the g-type lysozyme of Scophthalmus rhombus (94% overall identity). SmLysG possesses a Goose Egg White Lysozyme (GEWL) domain with conserved residues essential for catalytic activity. Recombinant SmLysG (rSmLysG) purified from yeast exhibits strong lysozyme activity against Micrococcus luteus. Enzyme assays showed that the optimal temperature and pH of rSmLysG are 30°C and pH 7.0, respectively. Substrate spectrum analysis indicated that rSmLysG inhibited the growth of a number of important fish pathogens of both Gram-negative and Gram-positive natures. SmLysG transcription was detected in multiple tissues and was upregulated in kidney and spleen by experimental challenges with lipopolysaccharide and bacterial pathogens that are, respectively, sensitive to and resistant against the lytic effect of rSmLysG. Comparative analysis showed that although bacterial infection also induced the expression of c-type lysozyme, the induction levels were much lower than those of SmLysG. Taken together, these results indicate that SmLysG is a functional g-type lysozyme with a wide working range and is involved in innate immune defense against general bacterial infection.

SmC2P1, a C2 domain protein from Scophthalmus maximus that binds bacterial pathogen-infected lymphocytes and reduces bacterial survival.

C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36-38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight beta-strands with a Ca(2+)-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection.

Application of a magnetotactic bacterium, Stenotrophomonas sp. to the removal of Au(III) from contaminated wastewater with a magnetic separator.

In this study, the feasibility of applying a magnetotactic bacterial isolate (MTB), Stenotrophomonas sp. to the removal of Au(III) was investigated. Biosorption experiments showed that Au(III) biosorption capacity exhibited no significant difference in the initial pH range of 1.0-5.5, while decreased more significantly in the initial pH range of 5.5-13.0. Langmuir isotherm indicated that the maximum Au(III) biosorption capacity of Stenotrophomonas sp. were 506, 369 and 308 mg g(-1) dry weight biomass at the initial pH values of 2.0, 7.0 and 12.0, respectively. Thiourea was proved to be an effective desorbent to recover Au from the MTB biomass and 91% Au adsorbed on the biomass could be recovered at equilibrium when the thiourea concentration was 0.8M. The magnetic separator developed by our research team used for separating Au loaded MTB biomass showed high separation efficiency, with 100% biomass removed at the magnetic intensity of 1200 Gs in 180 min. The analyses from FTIR and XRD further confirmed that the reduction of Au(III) to Au(0) by the reductants on the MTB biomass occurred, and the deposition of nano-crystal Au(0) particles, ranging from 24.7 to 31.4 nm, could be estimated on the biomass surface.

Biodegradation of high concentration phenol containing heavy metal ions by functional biofilm in bioelectro-reactor.

Functional microorganisms to high concentration phenol containing Cr6+ and Pb2+ were cultured and biofilm was formed on polypropylene packings in bioelectro-reactor. It was found that the biodegradation capability of such biofilm to phenol changed with the applied voltage. Under the optimal electric field conditions (voltage of 3.0 V, electric field of strength 17.7 V/m and current density of 1.98 A/m2), biodegradation efficiency of phenol aof concentration of 1200 mg/L increased 33% compared to the instance without applying electric field. However, voltage had inverse effect on biodegradation, as microorganisms were killed under strong electric field. Voltage had little effect on heavy ions elimination. Higher absorption rate of Cr6+ and Pb2+ was observed when changing pH from acidic to neutral. The experiment results indicated that, after treatment, 10 L phenol of 2400 mg/L was biodegraded completely within 55 h and concentrations of Cr6+ and Pb2+ dropped to less than 1 mg/L within 12 h and 6 h, from initial values of 50 mg/L and 30 mg/L, respectively.