Mahalingam Anjugam

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties

ABSTRACT The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp‐Lec). The Pp‐Lec was purified by affinity chromatography with mannose coupled sepharose CL‐4B column and it exhibits single band with a molecular weight of 155 kDa in SDS‐PAGE. The surface morphology of purified Pp‐Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X‐ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp‐Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp‐Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp‐Lec showed the broad spectrum of antibacterial activity against Gram‐positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp‐Lec against selective Gram‐negative bacteria showed the disruption of biofilm architecture at the concentration of 50 &mgr;g ml−1. HighlightsImmune molecule lectin was successfully purified from the blue swimmer crab Portunus pelagicus using mannose coupled sepharose CL‐4B column.Functional role of lectin in the immune system of P. pelagicus was noticed.Antibiofilm activity of lectin in destroying biofilm architecture of Gram negative bacteria was discussed.

Role of purified β-1, 3 glucan binding protein (β-GBP) from Paratelphusa hydrodromus and their anti-inflammatory, antioxidant and antibiofilm properties

Abstract &bgr;‐ 1, 3‐glucan binding protein (&bgr;‐GBP), a pattern recognition protein (PRP), plays a critical role in triggering the innate immune response by detecting &bgr;‐glucan found on the surface of microbes. In the present study, &bgr;‐GBP was purified from the haemolymph of rice field crab Paratelphusa hydrodromus by affinity column chromatography. The monomeric protein Ph‐&bgr;‐GBP appeared as a single band with a molecular weight of approximately 95 kDa in SDS‐PAGE analysis and its purity was determined to be 89% by HPLC. MALDI‐TOF/TOF analysis revealed that, the purified 95 kDa protein display 36% similarity with &bgr;‐GBP of crayfish Astacus lepidodactylus. Purified Ph‐&bgr;‐GBP exhibited increased agglutination, phagocytic activity and encapsulation in a dose‐dependent manner, indicating the involvement of Ph‐&bgr;‐GBP in cellular immune response against pathogens in crustaceans. Moreover, addition of Ph‐&bgr;‐GBP increased the prophenoloxidase (proPO) and serine protease activity, possibly contributing to the clearance of pathogens. The antioxidant activity of Ph‐&bgr;‐GBP was determined by DPPH radical scavenging activity demonstrates maximum scavenging activity of 78.4%. In addition, RBC membrane stabilization and inhibition of protein (albumin) denaturation proved anti‐inflammatory property of Ph‐&bgr;‐GBP. Furthermore, light microscopic and confocal laser scanning microscopic analysis revealed that the reactive compound (laminarin and Ph‐&bgr;‐GBP) reduced the biofilm thickness of Gram‐positive (Enterococcus faecalis) and Gram‐negative (Vibrio parahaemolyticus) bacteria at the concentration of 25 &mgr;g/ml. Taken together, our results demonstrate that, the &bgr;‐GBP triggers proPO activating system in rice field crab P. hydrodromus and plays a vital role in innate defense mechanism against invading pathogens. HighlightsImmune molecule &bgr;‐GBP was purified from the heamolymph of rice field crab Paratelphusa hydrodromus by Sepharose CL‐6B affinity column chromatography.The immunological role of Ph‐&bgr;‐GBP was proved through agglutination reaction, phagocytosis and encapsulation assays.Antioxidant and anti‐inflammation activity of Ph‐&bgr;‐GBP was explored.The antibiofilm potential of reactive compound was evidenced against Gram positive E. feacalis & Gram negative V. parahaemolyticus.

Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus

ABSTRACT The present study evaluated the dietary supplementation of probiotic Bacillus licheniformis Dahb1 on the growth performance, immune parameters and antioxidant enzymes activities in serum and mucus as well as resistance against Aeromonas hydrophila in Mozambique tilapia Oreochromis mossambicus. Fish (24±2.5g) were fed separately with three diets, 1) commercial diet (control), 2) diet containing probiotic at 105cfug−1 (D1) and 3) diet containing probiotic at 107cfug−1 (D2) for 4 weeks. Growth performance in term of final weight (FW) specific growth rate (SGR) and feed conversion ratio (FCR), immune parameters of total protein (TP), alkaline phosphatase (ALP), myeloperoxidase (MPO), lysozyme (LYZ), reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant parameters of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in serum and mucus were evaluated after 2nd and 4th weeks. The FW, SGR, and FCR of fish fed with D1 and D2 significantly improved (p<0.05). The activities of ALP, LYZ and MPO in the mucus were significantly higher (p<0.05) in fish that fed D1 and D2. The TP, ROS, RNS, SOD and GPx in the serum were significantly higher (p<0.05) in fish that fed D1 and D2. In addition, the challenge test showed that fish fed D1 and D2 enhanced significantly (p<0.05) the resistance against A. hydrophila (1×107cells ml−1). In conclusion, probiotic B. licheniformis Dahb1 can be applied in diet at 107cfug−1 to improve healthy status and resistance against A. hydrophila in tilapia farming. HIGHLIGHTSBacillus licheniformis Dahb1 could be used as feed additive to O. mossambicus.The growth performance of fish fed with the diet containing B. licheniformis Dahb1 significantly improved.Antioxidant and immune parameters in mucus and serum enhanced in B. licheniformis Dahb1 groups.Dietary supplementation of probiotic improved survival rate of O. mossambicus against A. hydrophila challenge.The optimal dose of dietary supplementation of B. licheniformis Dahb1 was 107cfug−1 diet.

A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus

Graphical abstract Figure. No Caption available. Highlights&bgr;‐1, 3 glucan binding protein was purified from Indian white shrimp Fenneropenaeus indicus.Through MALDI‐TOF/TOF analysis, the purified protein was identified as &bgr;‐GBP.The effect of T°, pH and different metallic ion concentrations on &bgr;‐GBP for phenoloxidase activation was evaluated.The phenoloxidase reaction product was analyzed for antibacterial and antibiofilm potential. ABSTRACT The present study reports the purification of novel immune molecule &bgr;‐1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fi&bgr;‐GBP). The purified Fi&bgr;‐GBP had 95 kDa molecular weight in SDS‐PAGE analysis. MALDI‐TOF/TOF analysis revealed that the purified Fi&bgr;‐GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for &bgr;‐1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e + 12 and 2.05e + 8, respectively. The phenoloxidase activity (PO) of Fi&bgr;‐GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fi&bgr;‐GBP had the specific binding site for soluble or insoluble &bgr;‐glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fi&bgr;‐GBP activating PO were 40 °C and pH 7–8. Moreover, even at 100 °C Fi&bgr;‐GBP enhanced PO activity highlighting that Fi&bgr;‐GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fi&bgr;‐GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from N&agr;−Benzoyl‐l‐Arginine 4‐Nitroanilide hydrochloride showed that serine protease activity was induced by Fi&bgr;‐GBP and also increased concentration of Fi&bgr;‐GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Effect of β-1, 3 glucan binding protein based zinc oxide nanoparticles supplemented diet on immune response and disease resistance in Oreochromis mossambicus against Aeromonas hydrophila

&NA; Recently, several immunostimulants such as &bgr;‐glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus &bgr;‐1, 3 glucan binding protein based zinc oxide nanoparticles (Pp&bgr;‐GBP‐ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune‐related protein &bgr;‐GBP was purified from the haemolymph of P. pelagicus using Sephadex G‐100 affinity column chromatography. Pp&bgr;‐GBP‐ZnO NPs was physico‐ chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control‐group I) and Pp&bgr;‐GBP (group II, III, IV), Pp&bgr;‐GBP‐ZnO NPs (group V, VI, VII), chem‐ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Pp&bgr;‐GBP‐ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Pp&bgr;‐GBP‐ZnO NPs supplemented diet than others. Antibiofilm potential of Pp&bgr;‐GBP‐ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 &mgr;m at the concentration of 50 &mgr;g/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml−1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Pp&bgr;‐GBP‐ZnO NPs. Thus our study suggests that dietary supplementation of Pp&bgr;‐GBP‐ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus. Graphical abstract Figure. No caption available. Highlights&bgr;‐ 1, 3 glucan binding protein based zinc oxide nanoparticles (Pp&bgr;‐GBP‐ZnO NPs) were synthesized and characterized.Pp&bgr;‐GBP‐ZnO NPs supplemented diet actively enhances the immune response of Oreochromis mossambicus.Pp&bgr;‐GBP‐ZnO NPs supplemented diet fed fishes exhibit resistance against Aeromonas hydrophila infection.

Multipurpose efficacy of ZnO nanoparticles coated by the crustacean immune molecule β-1, 3-glucan binding protein: Toxicity on HepG2 liver cancer cells and bacterial pathogens

The effective treatment of cancer and bacterial pathogens are two key challenges in modern nanomedicine. Here, zinc oxide nanoparticles (ZnO NPs) were fabricated using the crustacean immune molecule β-1, 3- glucan binding protein (Phβ-GBP, 100kDa) purified from the heamolymph of Paratelphusa hydrodromus. β-GBP coated zinc oxide nanoparticles (Phβ-GBP-ZnO NPs) were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high resolution-transmission electron microscopy (HR-TEM) analyses. Phβ-GBP-ZnO NPs inhibited the growth of Staphylococcus aureus and Proteus vulgaris. Protein and nucleic acid leakage assays showed that Phβ-GBP-ZnO NPs facilitate membrane permeability leading to cell death. The antibacterial activity of Phβ-GBP-ZnO NPs was due to the high level of reactive oxygen species (ROS) release from bacterial cells post-treatment with 75μg/mL of Phβ-GBP-ZnO NPs. Confocal laser scanning microscopy pointed out that biofilm thickness was highly reduced post-treatment with nanoparticles. Cytotoxicity on human liver carcinoma (HepG2) cells highlighted that 75μg/mL of Phβ-GBP-ZnO NPs inhibited viability of HepG2 cells. Phase contrast microscopy showed key morphological changes of HepG2 cells post-treatment with Phβ-GBP-ZnO NPs. Overall, Phβ-GBP-ZnO NPs can be further considered for the development of novel drugs against microbial pathogens and HepG2 cells.

Antibiofilm Competency of Portunus pelagicus Haemolymph and Identificationof its Bioactive Compounds

The marine atmosphere may be investigated as a rich source for novel drugs. In past decades, a number of marine-derived compounds have been isolated and identified. The current effort was made on haemolymph of blue swimmer crab Portunus pelagicus to mark its antimicrobial activity against eight pathogenic bacteria of Gram positive, Gram negative and fungus Candida albicans. Agar well diffusion method was put into practice in deliverance of antibacterial and antifungal activity by measuring zone of inhibition. The findings displays, P. pelagicus haemolymph at the concentration of 150 μl can effectively act against all of challenged bacteria and fungus. Besides, the antibiofilm property of haemolymph against selected bacteria and fungus C. albicans which revealed the potential bactericidal and fungicidal effect at 150 μl concentration of haemolymph. Moreover, the antimicrobial and antibiofilm potent of crab haemolymph was confirmed by growth curve analysis, biofilm growth inhibition and protein leakage assay which collectively conclude the ability of haemolymph in microbial growth inhibition. For the first time, the bioactive compounds were screened from the haemolymph of P. pelagicus through Gas chromatography- Mass spectrometry analysis discloses the existence of 15 types of compounds in haemolymph being in charge for antimicrobial activity.

Searching for crab-borne antimicrobial peptides: Crustin from Portunus pelagicus triggers biofilm inhibition and immune responses of Artemia salina against GFP tagged Vibrio parahaemolyticus Dahv2

Graphical abstract Figure. No caption available. HighlightsPurification of crustin (Pp‐Cru) from Portunus pelagicus was characterized by HPLC, CD, XRD, and FTIR analyses.We reported an enhancement of the immune system by Pp‐Cru through in vivo and in vitro analysis.Synergic antibacterial and antibiofilm properties of Pp‐Cru were observed against key bacterial pathogens.Live and dead assay showed ultra‐low bacterial cell viability post‐treatment with Pp‐Cru.In vivo study of crustin was assessed with Artemia salina as a model crustacean. &NA; Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL‐6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS‐PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp‐Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp‐Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp‐Cru on Staphylococcus aureus, Enterococcus faecalis (Gram‐ positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram‐negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 &mgr;g/ml of Pp‐Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp‐Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp‐Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture.

Phenoloxidase activation, antimicrobial, and antibiofilm properties of β-glucan binding protein from Scylla serrata crab hemolymph

In this study, we purified β-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata β-GBP (Ss-β-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with β-GBP of Astacus leptodactylus. Ss-β-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-β-GBP. The purified Ss-β-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-β-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-β-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60μg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-β-GBP at 50 and 100μg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-β-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-β-GBP might be widely used to suppress the growth of pathogenic bacteria.

β-1, 3 glucan binding protein based selenium nanowire enhances the immune status of Cyprinus carpio and protection against Aeromonas hydrophila infection

Abstract In the present study, immunoenhancing effect of &bgr;‐1, 3 glucan binding protein based selenium nanowire (Ph&bgr;‐GBP‐SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule &bgr;‐GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Ph&bgr;‐GBP‐SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30–50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Ph&bgr;‐GBP, sodium selenite, selenomethionine and Ph&bgr;‐GBP‐SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Ph&bgr;‐GBP‐SeNWs supplemented diet were significantly increased in dose‐dependent manner, which was observed at two different interval period (15th and 30th day). Also, Ph&bgr;‐GBP‐SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Ph&bgr;‐GBP‐SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Ph&bgr;‐GBP‐SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Ph&bgr;‐GBP‐SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens. Graphical abstract Figure. No Caption available. Highlights&bgr;‐GBP was purified from the Paratelphusa hydrodromus haemolymph.Ph&bgr;‐GBP based selenium nanowires (Ph&bgr;‐GBP SeNWs) were synthesized and characterized.Ph&bgr;‐GBP SeNWs supplemented diet and its impact on immune parameters were studied.