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  1. Arockiaraj J, Chaurasia MK, Kumaresan V, Palanisamy R, Harikrishnan R, Pasupuleti M, et al.
    Fish Shellfish Immunol, 2015 Apr;43(2):364-74.
    PMID: 25575476 DOI: 10.1016/j.fsi.2014.12.036
    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.
  2. Arockiaraj J, Kumaresan V, Bhatt P, Palanisamy R, Gnanam AJ, Pasupuleti M, et al.
    Peptides, 2014 Mar;53:79-88.
    PMID: 24269604 DOI: 10.1016/j.peptides.2013.11.008
    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 β-sheet confirmation. The β-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.
  3. Chaurasia MK, Palanisamy R, Bhatt P, Kumaresan V, Gnanam AJ, Pasupuleti M, et al.
    Microbiol Res, 2015 Jan;170:78-86.
    PMID: 25271126 DOI: 10.1016/j.micres.2014.08.011
    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.
  4. Bhatt P, Chaurasia MK, Palanisamy R, Kumaresan V, Arasu A, Sathyamoorthi A, et al.
    Fish Shellfish Immunol, 2014 Aug;39(2):245-53.
    PMID: 24861891 DOI: 10.1016/j.fsi.2014.05.019
    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.
  5. Arasu A, Kumaresan V, Sathyamoorthi A, Chaurasia MK, Bhatt P, Gnanam AJ, et al.
    Microbiol Res, 2014 Nov;169(11):824-34.
    PMID: 24780642 DOI: 10.1016/j.micres.2014.03.005
    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.
  6. Arockiaraj J, Bhatt P, Kumaresan V, Dhayanithi NB, Arshad A, Harikrishnan R, et al.
    Fish Shellfish Immunol, 2015 Nov;47(1):221-30.
    PMID: 26363233 DOI: 10.1016/j.fsi.2015.09.015
    In this study, we reported a molecular characterization of three CC chemokines namely, CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 which are were identified from the established cDNA library of striped murrel Channa striatus. Multiple sequence alignment of all the three chemokines revealed the presence of gene specific domains and motifs including small cytokine domain, IL8 like domain, receptor binding site and glycosaminoglycan (GAG) binding sites. Three dimensional structures of the chemokines under study showed an important facet on their anti-microbial property. Tissue specific mRNA expression showed that the CsCC-Chem14 is highly expressed in spleen, CsCC-Chem20 in liver and CsCC-Chem25 in trunk kidney. On challenge C. striatus with oomycete fungus Aphanomyces invadans, both CsCC-Chem20 and CsCC-Chem25 showed significant (P < 0.05) up-regulation compared to CsCC-Chem14. The increase in the expression levels of CsCC-Chem20 and CsCC-Chem25 due to infection showed that they are antimicrobial proteins. But considering the CsCC-Chem14 expression, it is found to be a constitutive chemokine and is involved in homeostatic function in spleen of C. striatus. C. striatus challenged with bacteria Aeromonas hydrophila also exhibited different up-regulation pattern in all the three chemokines at various time points. However, extensive studies are required to determine the functional activities of CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 in vitro and in vivo to gain more knowledge at the molecular and proteomic levels.
  7. Kumaresan V, Bhatt P, Ganesh MR, Harikrishnan R, Arasu M, Al-Dhabi NA, et al.
    Mol Immunol, 2015 Dec;68(2 Pt B):421-33.
    PMID: 26477736 DOI: 10.1016/j.molimm.2015.10.001
    In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.
  8. Kumaresan V, Pasupuleti M, Arasu MV, Al-Dhabi NA, Arshad A, Amin SMN, et al.
    Mol Biol Rep, 2018 Dec;45(6):2511-2523.
    PMID: 30306509 DOI: 10.1007/s11033-018-4418-y
    Snakehead murrel, Channa striatus is an economically important aquatic species in Asia and are widely cultured and captured because of its nutritious and medicinal values. Their growth is predominantly affected by epizootic ulcerative syndrome (EUS) which is primarily caused by an oomycete fungus, Aphanomyces invadans. However, the molecular mechanism of immune response in murrel against this infection is still not clear. In this study, transcriptome technique was used to understand the molecular changes involved in C. striatus during A. invadans infection. RNA from the control (CF) and infected fish (IF) groups were sequenced using Illumina Hi-seq sequencing technology. For control group, 28,952,608 clean reads were generated and de novo assembly was performed to produce 60,753 contigs. For fungus infected group, 25,470,920 clean reads were obtained and assembled to produce 58,654 contigs. Differential gene expression analysis revealed that a total of 146 genes were up-regulated and 486 genes were down regulated. Most of the differentially expressed genes were involved in innate immune mechanism such as pathogen recognition, signalling and antimicrobial mechanisms. Interestingly, few adaptive immune genes, especially immunoglobulins were also significantly up regulated during fungal infection. Also, the results were validated by qRT-PCR analysis. These results indicated the involvement of various immune genes involved in both innate and adaptive immune mechanism during fungal infection in C. striatus which provide new insights into murrel immune mechanisms against A. invadans.
  9. Ravichandran G, Kumaresan V, Mahesh A, Dhayalan A, Arshad A, Arasu MV, et al.
    Int J Biol Macromol, 2018 Jan;106:1014-1022.
    PMID: 28837852 DOI: 10.1016/j.ijbiomac.2017.08.098
    Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.
  10. Bhatt P, Kumaresan V, Palanisamy R, Ravichandran G, Mala K, Amin SMN, et al.
    Fish Shellfish Immunol, 2018 Jan;72:670-678.
    PMID: 29162541 DOI: 10.1016/j.fsi.2017.11.036
    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.
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