A mini review on immune role of chemokines and its receptors in snakehead murrel Channa striatus.

Chemokines are ubiquitous cytokine molecules involved in migration of cells during inflammation and normal physiological processes. Though the study on chemokines in mammalian species like humans have been extensively studied, characterization of chemokines in teleost fishes is still in the early stage. The present review provides an overview of chemokines and its receptors in a teleost fish, Channa striatus. C. striatus is an air breathing freshwater carnivore, which has enormous economic importance. This species is affected by an oomycete fungus, Aphanomyces invadans and a Gram negative bacteria Aeromonas hydrophila is known to cause secondary infection. These pathogens impose immune changes in the host organism, which in turn mounts several immune responses. Of these, the role of cytokines in the immune response is immense, due to their involvement in several activities of inflammation such as cell trafficking to the site of inflammation and antigen presentation. Given that importance, chemokines in fishes do have significant role in the immunological and other physiological functions of the organism, hence there is a need to understand the characteristics, activities and performace of these small molecules in details.

Molecular profiles and pathogen-induced transcriptional responses of prawn B cell lymphoma-2 related ovarian killer protein (BOK).

In this study, we have reported a molecular characterization of the first B cell lymphoma-2 (BCL-2) related ovarian killer protein (BOK) from freshwater prawn Macrobrachium rosenbergii (Mr). BOK is a novel pro-apoptotic protein of the BCL-2 family that entails in mediating apoptosis to remove cancer cells. A cDNA sequence of MrBOK was identified from the prawn cDNA library and its full length was obtained by internal sequencing. The coding region of MrBOK yields a polypeptide of 291 amino acids. The analysis revealed that MrBOK contains a transmembrane helix at V(261)-L(283) and a putative BCL-2 family domain at V(144)-W(245). MrBOK also possessed four putative BCL-2 homology domains including BH1, BH2, BH3 and weak BH4. The BH3 contains 21 binding sites and among them five residues are highly conserved with the aligned BOK proteins. The homology analysis showed that MrBOK shared maximum similarity with the Caligus rogercresseyi BOK A. The topology of the phylogenetic tree was classified into nine sister groups which includes BOK, BAK, BAX, BAD, BCL-2, BCL-XL, NR13 and MCL members. The BOK protein group further sub-grouped into vertebrate and invertebrate BOK, wherein MrBOK located within insect monophyletic clad of invertebrate BOK. The secondary structural analysis showed that MrBOK contains 11 α-helices (52.2%) which are connected over random coils (47.7%). The 3D structure of MrBOK showed three central helices (α6, α7 and α8) which formed the core of the protein and are flanked on one side by α1, α2 and α3, and on the other side by α4, α5 and α11. MrBOK mRNA is expressed most abundantly (P < 0.05) in ovary compared to other tissues taken for analysis. Hence ovary was selected to study the possible roles of MrBOK mRNA regulation upon bacterial (Aeromonas hydrophila and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodovirus] infection. During bacterial and viral infection, the highest MrBOK mRNA transcription was varied at different time points. In bacterial infected ovary tissue, the highest mRNA expression was at 24 h post-infection, whereas in viral infection, the expression was highest at 48 h post-infection. Thus we can conclude that MrBOK functions as an apoptotic protein in intracellular programmed cell-death pathway to counteract the anti-apoptotic proteins released by bacterial and viral pathogens at the time of infection. This is the first study that emphasizes the importance of BOK during bacterial and viral infection in crustacean.

Macrobrachium rosenbergii mannose binding lectin: synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis.

Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii (Mr). Two short peptides (MrMBL-N20: (20)AWNTYDYMKREHSLVKPYQG(39) and MrMBL-C16: (307)GGLFYVKHKEQQRKRF(322)) were synthesized from the MrMBL polypeptide. The purity of the MrMBL-N20 (89%) and MrMBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. MrMBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the MrMBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, MrMBL-C16 peptide produced the highest activity compared to the MrMBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The MrMBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the MrMBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which is involved in antimicrobial mechanism. Moreover, MrMBL derived MrMBL-N20 and MrMBL-C16 peptides are important antimicrobial peptides for the recognition and eradication of viral and bacterial pathogens.

Molecular cloning, characterization and gene expression of murrel CXC chemokine receptor 3a against sodium nitrite acute toxicity and microbial pathogens.

CXCR3 is a CXC chemokine receptor 3 which binds to CXC ligand 4 (CXCL4), 9, 10 and 11. CXC chemokine receptor 3a (CXCR3a) is one of the splice variants of CXCR3. It plays crucial role in defense and other physiological processes. In this study, we report the molecular cloning, characterization and gene expression of CXCR3a from striped murrel Channa striatus (Cs). The full length CsCXCR3a cDNA sequence was obtained from the constructed cDNA library of striped murrel by cloning and sequencing using an internal sequencing primer. The full length sequence is 1425 nucleotides in length including an open reading frame of 1086 nucleotides which is encoded with a polypeptide of 361 amino acids (mol. wt. 40 kDa). CsCXCR3a domain analysis showed that the protein contains a G protein coupled receptor between 55 and 305 along with its family signature at 129-145. The transmembrane prediction analysis showed that CsCXCR3a protein contains 7 transmembrane helical regions at 34-65, 80-106, 113-146, 154-181, 208-242, 249-278 and 284-308. The 'DRY' motif from CsCXCR3a protein sequence at (140)Asp-(141)Arg-(142)Tyr which is responsible for G-protein binding is also highly conserved with CXCR3 from other species. Phylogenetic tree showed that the CXC chemokine receptors 3, 4, 5 and 6, each formed a separate clade, but 1 and 2 were clustered together, which may be due to the high similarity between these receptors. The predicted 3D structure revealed cysteine residues, which are responsible for 'CXC' motif at 116 and 198. The CsCXR3a transcript was found to be high in kidney, further its expression was up-regulated by sodium nitrite acute toxicity exposure, fungal, bacterial and poly I:C challenges. Overall, these results supported the active involvement of CsCXCR3a in inflammatory process of striped murrel during infection. However, further study is necessary to explore the striped murrel chemokine signaling pathways and their roles in defense system.

A redox active site containing murrel cytosolic thioredoxin: analysis of immunological properties.

In this study, we have reported the immunological properties of cDNA encoding thioredoxin which is obtained from the database of Channa striatus (named as CsTRx) cDNA library. The analysis showed that the CsTRx polypeptide contains a thioredoxin domain between Val(2) and Asn(106). The domain possessed a thioredoxin active family at 24­42 along with a redox active site (also known as catalytic center) at (31)WCGPC(35). The analysis showed that the catalytic center is responsible for the control of protein function. Phylogenetic study showed that CsTRx clustered together with vertebrate TRx-1. Based on the phylogenetic analysis and other bioinformatics analysis, it is confirmed that the characterized CsTRx belongs to TRx-1 family. In addition, the sub-cellular localization prediction analysis showed that CsTRx is a cytosol thioredoxin. The highest gene expression was observed in gill (P < 0.05). Further, its transcriptional modulation was evaluated under fungal (Aphanomyces invadans), bacterial (Aeromonas hydrophila) and H2O2 challenges. The recombinant CsTRx protein was over-expressed and purified using an Escherichia coli expression vector system. We conducted a H2O2 peroxidase assay using recombinant CsTRx protein under various pH and temperature. Further, we studied the influence of recombinant CsTRx protein on C. striatus spleen leukocyte activation. The recombinant CsTRx protein enhanced the cell proliferation in a concentration dependant manner. The results of antioxidant analysis showed that the antioxidant capacity of recombinant CsTRx protein was determined to be 4.2 U/mg protein. We conducted an insulin disulfides assay to study the enzymatic oxidoreductase activity of CsTRx and we observed no activity in the control group. But the recombinant CsTRx protein addition rapidly increased the enzymatic oxidoreductase activity. Over all, the results showed that the CsTRx may contain potential antioxidant properties, which could regulate the oxidative stress created by various biological pathogens as well as chemical stress in the immune system of C. striatus, thus protecting it.

A murrel interferon regulatory factor-1: molecular characterization, gene expression and cell protection activity.

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27% α-helices (85 aa residues), 5.7% ß-sheets (19 aa residues) and 67.19% random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (-284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.

A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression.

We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region, CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28-109), and in C-terminal region, it carries another SOD Fe domain (114-220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96%). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 α-helices (52.4%), 3 ß-sheets (8.8%) and 38.8% random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P < 0.05) changes in expression when compared to their non-injected and fish physiological saline-injected controls. The SOD activity shows that the activity increases with the spread of infection and decreases once the molecule controls the pathogen. The capacity of superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level.

Role of Type 4B Secretion System Protein, IcmE, in the Pathogenesis of Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever, a life-threatening zoonotic disease. C. burnetii replicates within an acidified parasitophorous vacuole derived from the host lysosome. The ability of C. burnetii to replicate and achieve successful intracellular life in the cell cytosol is vastly dependent on the Dot/Icm type 4B secretion system (T4SSB). Although several T4SSB effector proteins have been shown to be important for C. burnetii virulence and intracellular replication, the role of the icmE protein in the host-C. burnetii interaction has not been investigated. In this study, we generated a C. burnetii Nine Mile Phase II (NMII) mutant library and identified 146 transposon mutants with a single transposon insertion. Transposon mutagenesis screening revealed that disruption of icmE gene resulted in the attenuation of C. burnetii NMII virulence in SCID mice. ELISA analysis indicated that the levels of pro-inflammatory cytokines, including interleukin-1ß, IFN-γ, TNF-α, and IL-12p70, in serum from Tn::icmE mutant-infected SCID mice were significantly lower than those in serum from wild-type (WT) NMII-infected mice. Additionally, Tn::icmE mutant bacteria were unable to replicate in mouse bone marrow-derived macrophages (MBMDM) and human macrophage-like cells (THP-1). Immunoblotting results showed that the Tn::icmE mutant failed to activate inflammasome components such as IL-1ß, caspase 1, and gasdermin-D in THP-1 macrophages. Collectively, these results suggest that the icmE protein may play a vital role in C. burnetii virulence, intracellular replication, and activation of inflammasome mediators during NMII infection.

An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: analysis of immune properties.

In this study, we have reported the first histone characterized at molecular level from freshwater prawn Macrobrachium rosenbergii (MrHis). A full length cDNA of MrHis (751 base pairs) was identified from an established M. rosenbergii cDNA library using GS-FLX technique. It encodes 137 amino acid residues with a calculated molecular mass of 15 kDa and an isoelectric point of 10.5. MrHis peptide contains a histone H2A signature between 21 and 27 amino acids. Homologous analysis showed that MrHis had a significant sequence identity (99%) with other known histone H2A groups especially from Penaeus monodon. Phylogenetic analysis of MrHis showed a strong relationship with other amino acid sequences from histone H2A arthropod groups. Further phylogenetic analysis showed that the MrHis belongs to histone H2A superfamily and H2A1A sub-family. Secondary structure of MrHis showed that the protein contains 50.36% α-helical region and 49.64% coils. The 3D model of MrHis was predicted by I-Tasser program and the model was evaluated for quality analysis including C-score analysis, Ramachandran plot analysis and RMSD analysis. The surface view analysis of MrHis showed the active domain at the N terminal. The antimicrobial property of MrHis protein was confirmed by the helical structure and the total hydrophobic surface along with its net charge. The MFE of the predicted RNA structure of MrHis is -128.62 kcal/mol, shows its mRNA stability. Schiffer-Edmundson helical wheel analysis of the N-terminal of MrHis showed a perfect amphipathic nature of the peptide. Significantly (P < 0.05) highest gene expression was noticed in the hemocyte and is induced with viral (WSBV and MrNV) and bacteria (A eromonas hydrophila and Vibrio harveyi) infections. The coding sequence of recombinant MrHis protein was expressed in a pMAL vector and purified to study the antimicrobial properties. The recombinant product showed antimicrobial activity against both Gram negative and Gram positive bacteria. In this study, the recombinant MrHis protein displayed antimicrobial activity in its entirety. Hence, it is possible to suggest that the activity may be due to the direct defense role of histone or its N-terminal antimicrobial property. However, this remains to be verified by detailed investigations.

Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin.

Antimicrobial peptides (AMPs) are innate molecules that are found in a wide variety of species ranging from bacteria to humans. In recent years, excessive usage of antibiotics resulted in development of multi-drug resistant pathogens which made researchers to focus on AMPs as potential substitute for antibiotics. Lily type mannose-binding lectin is an extended super-family of structurally and evolutionarily related sugar binding proteins. These lectins are well-known AMPs which play important roles in fish defense mechanism. Here, we report a full-length lily type lectin-2 (LTL-2) identified from the cDNA library of striped murrel, Channa striatus (Cs). CsLTL-2 protein contained B-lectin domain along with three carbohydrate binding sites which is a prominent characteristic functional feature of LTL. The mRNA transcripts of CsLTL-2 were predominantly expressed in gills and considerably up-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila). To evaluate the antimicrobial activity of the carbohydrate binding region of CsLTL-2, the region was synthesized (QP13) and its bactericidal activity was analyzed. In addition, QP13 was labeled with fluorescein isothiocyanate (FITC) and its binding affinity with the bacterial cell membranes was analyzed. Minimum inhibitory concentration assay revealed that QP13 inhibited the growth of Escherichia coli at a concentration of 80 µM/ml. Confocal microscopic observation showed that FITC tagged QP13 specifically bound to the bacterial membrane. Fluorescence assisted cell sorter (FACS) assay showed that QP13 reduced the bacterial cell count drastically. Therefore, the mechanism of action of QP13 on E. coli cells was determined by propidium iodide internalization assay which confirmed that QP13 induced bacterial membrane disruption. Moreover, the peptide did not show any cytotoxicity towards fish peripheral blood leucocytes. Taken together, these results support the potentiality of QP13 that can be used as an antimicrobial agent against the tested pathogens.

Functional roles and gene regulation of tumor necrosis factor receptor 1 in freshwater striped murrel.

In this study, a complete molecular characterization of tumor necrosis factor receptor 1 (TNFR1) which was identified from the constructed cDNA library of striped murrel Channa striatus (Cs) is reported. The CsTNFR1 encoded a type I membrane receptor protein that contains 399 amino acids including three cysteine-rich domains (CRDs) at CRD1(41-46), CRD2(79-118) and CRD3(120-159) in the extracellular region and a putative TNF receptor-associated factor (TRAF) site at 245-253 and a death domain between 297 and 388 in the cytoplasmic region which is essential for induction of apoptosis. The predicted molecular mass of CsTNFR1 is 45kDa and its corresponding theoretical isoelectric point (pI) is 6.3. CsTNFR1 shared maximum identity with TNFR1 from olive flounder Paralichthys olivaceus (82%). Real-time PCR showed that CsTNFR1 gene was expressed most abundantly (P<0.05) in the head kidney. Further, CsTNFR1 mRNA transcription was studied after challenge with fungus Apanomyces invadans and bacteria Aeromonas hydrophila. The fungus injected murrels showed a highest expression at 48h, whereas bacteria injected murrels showed at 24h. The gene expression studies revealed that CsTNFR1 may be involved in innate immune process of murrels against pathogenic infections. The over-expressed and purified recombinant CsTNFR1 protein (rCsTNFR1) was subjected to TNF-α inhibition assay to confirm their specificity and activity against TNF-α which confirmed that the rCsTNFR1 inhibits the activity of TNF-α in a dose dependent manner where maximum inhibition (97%) was observed at 10,000 fold concentration of rCsTNFR1. In addition, the direct cytotoxic effect of rCsTNFR1 was analyzed against head kidney phagocyte. The results showed that the recombinant CsTNFR1 protein does not exhibit any significant cytotoxicity against head kidney phagocyte cells even at higher concentration (8µg/ml). Moreover, the recombinant protein was analyzed for respiratory burst activity in the presence of two different ROS inducers, opsonized zymosan (fungal cell wall component) and phorbol 12-myristate 13-acetate (PMA). The findings showed that the C. striatus head kidney phagocyte exposed to purified recombinant CsTNFR1 protein alone do not produced any ROS. However, opsonized zymosan induced recombinant CsTNFR1 protein significantly (P<0.05) enhanced the ROS production on concentration basis which is revealed that the ROS production depends on the concentration of the recombinant CsTNFR1 protein. Overall, the study showed that the CsTNFR1 plays an important role in the pathogen-induced inflammatory process of striped murrel.

An anti-apoptotic B-cell lymphoma-2 (BCL-2) from Channa striatus: Sequence analysis and delayed and advanced gene expression in response to fungal, bacterial and poly I:C induction.

B-cell lymphoma-2 (BCL-2) is a suppressor of apoptosis and inhibits the caspase dependent apoptosis pathway. In this study, we report molecular characterization of a cDNA sequence encoded of BCL-2 from striped murrel, Channa striatus. A partial cDNA sequence of CsBCL-2 was identified from the striped murrel cDNA library during annotation. Subsequently, the full length CsBCL-2 cDNA sequence was obtained by an internal sequencing method using a forward primer. The sequence contains 699 nucleotide base pairs which encode 232 amino acid residues. The domain and motif analysis revealed that the CsBCL-2 polypeptide consists of BCL-2 homologous domain BH4 at the N-terminal region between 4 and 21 and the BCL-2 homologous domains BH1, BH2 and BH3 between 87 and 187. The CsBCL-2 polypeptide sequence does not have a signal peptide region, but it consists of two novel transmembrane regions at 134-152 and 209-226. The sequence analysis showed that the CsBCL-2 has highest sequence identity (70%) with BCL-2 like protein 1 (BCL-2 L1) from pufferfish Takifugu rubripes. The phylogenetic analysis showed that the CsBCL-2 was situated in the BCL-2 L1 fish clade. The secondary analysis showed that the CsBCL-2 protein consists of 132 amino acid residues in the α-helical region and 100 amino acid residues in the random coil region. The validated 3D structure of CsBCL-2 showed the active residues Gly(135) and Arg(136) in the 7th α-helical position, whereas Trp(178) is in the 9th α-helical region. CsBCL-2 mRNA transcription is predominately present in spleen and is upregulated upon being induced with fungus Aphanomyces invadans, bacteria Aeromonas hydrophila, Escherichia coli LPS, Laminaria digitata beta-1,3-glucan and poly I:C. Overall, the CsBCL-2 mRNA transcription results indicate the potential involvement of CsBCL-2 in immune system of C. striatus. However, further research at proteomic level is necessary to examine these predictions.

A prawn core histone 4: derivation of N- and C-terminal peptides and their antimicrobial properties, molecular characterization and mRNA transcription.

This study investigates the complete molecular characterization including bioinformatics characterization, gene expression, synthesis of N and C terminal peptides and their antimicrobial activity of the core histone 4 (H4) from freshwater giant prawn Macrobrachium rosenbergii (Mr). A cDNA encoding MrH4 was identified from the constructed cDNA library of M. rosenbergii during screening and the sequence was obtained using internal sequencing primers. The MrH4 coding region possesses a polypeptide of 103 amino acids with a calculated molecular weight of 11kDa and an isoelectric point of 11.5. The bioinformatics analysis showed that the MrH4 polypeptide contains a H4 signature at (15)GAKRH(19). Multiple sequence alignment of MrH4 showed that the N-terminal (21-42) and C-terminal (87-101) antimicrobial peptide regions and the pentapeptide or H4 signature (15-19) are highly conserved including in humans. The phylogenetic tree formed two separate clades of vertebrate and invertebrate H4, wherein MrH4 was located within the arthropod monophyletic clade of invertebrate H4 groups. Three-dimensional model of MrH4 was established using I-TASSER program and the model was validated using Ramachandran plot analysis. Schiffer-Edmundson helical wheel modeling was used to predict the helix propensity of N (21-42) and C (87-101) terminal derived Mr peptides. The highest gene expression was observed in gills and is induced by viral [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacterial (Aeromonas hydrophila and Vibrio harveyi) infections. The N and C terminal peptides were synthesized and their antimicrobial and hemolytic properties were examined. Both peptides showed activity against the tested Gram negative and Gram positive bacteria; however, the highest activity was noticed against Gram negative bacteria. Among the two peptides used in this study, C-terminal peptide yielded better results than the N-terminal peptide. Therefore, C terminal peptide can be recommended for the development of an antimicrobial agent.

Immunological role of C4 CC chemokine-1 from snakehead murrel Channa striatus.

In this study, we have reported a cDNA sequence of C4 CC chemokine identified from snakehead murrel (also known as striped murrel) Channa striatus (named as CsCC-Chem-1) normalized cDNA library constructed by Genome Sequencing FLX™ Technology (GS-FLX™). CsCC-Chem-1 is 641 base pairs (bp) long that contain 438 bp open reading frame (ORF). The ORF encodes a polypeptide of 146 amino acids with a molecular mass of 15 kDa. The polypeptide contains a small cytokine domain at 30-88. The domain carries the CC motif at Cys(33)-Cys(34). In addition, CsCC-Chem-1 consists of another two cysteine residues at C(59) and C(73), which, together with C(33) and C(34), make CsCC-Chem-1 as a C4-CC chemokine. CsCC-Chem-1 also contains a 'TCCT' motif at 32-35 as CC signature motif; this new motif may represent new characteristic features, which may lead to some unknown function that needs to be further focused on. Phylogenitically, CsCC-Chem-1 clustered together with CC-Chem-1 from rock bream Oplegnathus fasciatus and European sea bass Dicentrarchus labrax. Significantly (P<0.05) highest gene expression was noticed in spleen and is up-regulated upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and virus (poly I:C) infection at various time points. The gene expression results indicate the influence of CsCC-Chem-1 in the immune system of murrel. Overall, the gene expression study showed that the CsCC-Chem-1 is a capable gene to increase the cellular response against various microbial infections. Further, we cloned the coding sequence of CsCC-Chem-1 in pMAL vector and purified the recombinant protein to study the functional properties. The cell proliferation activity of recombinant CsCC-Chem-1 protein showed a significant metabolic activity in a concentration dependent manner. Moreover, the chemotaxis assay showed the capability of recombinant CsCC-Chem-1 protein which can induce the migration of spleen leukocytes in C. striatus. However, this remains to be verified further at molecular and proteomic level.

Molecular characterization of a novel proto-type antimicrobial protein galectin-1 from striped murrel.

In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4-135. The domain carries a sugar binding site at 45-74 along with its signatures (H(45)-X-Asn(47)-X-Arg(49) and Trp(69)-X-X-Glu(72)-X-Arg(74)). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4µg/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25mM(-1) and d-glucose and d-fructose at 100mM(-1). The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gram-negative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.

A novel single-domain peptide, anti-LPS factor from prawn: synthesis of peptide, antimicrobial properties and complete molecular characterization.

In this study, we reported a complete molecular characterization including bioinformatics features, gene expression, peptide synthesis and its antimicrobial activities of an anti-lipopolysaccharide (LPS) factor (ALF) cDNA identified from the established cDNA library of freshwater prawn Macrobrachium rosenbergii (named as MrALF). The mature protein has an estimated molecular weight of 11.240 kDa with an isoelectric point of 9.46. The bioinformatics analysis showed that the MrALF contains an antimicrobial peptide (AMP) region between T54 and P77 with two conserved cysteine residues (Cys55 and Cys76) which have an anti-parallel ß-sheet confirmation. The ß-sheet is predicted as cationic with hydrophobic nature containing a net charge of +5. The depicted AMP region is determined to be amphipathic with a predicted hydrophobic face 'FPVFI'. A highest MrALF gene expression was observed in hemocytes and is up-regulated with virus [white spot syndrome baculovirus (WSBV)], bacteria (Aeromonas hydrophila) and Escherichia coli LPS at various time points. The LPS binding region of MrALF peptide was synthesized to study the antimicrobial property, bactericidal efficiency and hemolytic capacity. The peptide showed antimicrobial activity against both the Gram-negative and Gram-positive bacteria. The bactericidal assay showed that the peptide recognized the LPS of bacterial cell walls and binding on its substrate and thereby efficiently distinguishing the pathogens. The hemolytic activity of MrALF peptide is functioning in a concentration dependant manner. In summary, the comprehensive analysis of MrALF showed it to be an effective antimicrobial peptide and thus it plays a crucial role in the defense mechanism of M. rosenbergii.

A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties.

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P<0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.

A novel prophenoloxidase, hemocyanin encoded copper containing active enzyme from prawn: gene characterization.

The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended ß-sheets and a very few ß-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended ß-sheets and ß-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral and bacterial infected prawns. The highest enzyme activity was observed in hepatopancreas, which was also significantly higher (P<0.05) than detected in other tissues. Similar to gene expression results, the enzyme activity reached the peak at 24h p.i. and then the activity started decreasing. Overall results indicate that MrProPO is very likely to participate in the acute response against pathogen entry in prawns.

Fish lily type lectin-1 contains ß-prism architecture: immunological characterization.

In this study we report a full-length lily type lectin-1 (CsLTL-1) identified from striped murrel, Channa striatus. CsLTL-1 was identified from the established C. striatus cDNA library using GS-FLX™ genome sequencing technology and was found to contain 354 nucleotide base pairs and its open reading frame (ORF) encodes a 118 amino acid residue. CsLTL-1 mRNA is predominately expressed in the gills and is up-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila). Hemagglutination studies with recombinant CsLTL-1 show that, at 4µg/ml agglutinates occurs in a calcium independent manner and is inhibited in the presence of d-mannose (50mM) and d-glucose (100mM). The CsLTL-1 sequence was completely characterized using various bioinformatics tools. CsLTL-1 peptide contains a mannose binding site at 30-99 along with its specific motif of ß-prism architecture. The phylogenetic analysis showed that CsLTL-1 clustered together with LTL-1 from Oplegnathus fasciatus. CsLTL-1 protein 3D structure was predicted by I-Tasser program and the model was evaluated using Ramachanran plot analysis. The secondary structure analysis of CsLTL-1 reveals that the protein contains 23% ß-sheets and 77% coils. The overall results showed that CsLTL-1 is an important immune gene involved in the recognition and elimination of pathogens in murrels.

A prawn transglutaminase: molecular characterization and biochemical properties.

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg(180)-Gly(181)-Asp(182) and Arg(269)-Gly(270)-Asp(271). The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.