Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon.

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Antibiofilm and anticancer potential of ß-glucan-binding protein-encrusted zinc oxide nanoparticles.

ß-Glucan-binding protein (ßGBP) is important for the rational expansion of molecular biology. Here, zinc oxide nanoparticle (ZnONP) was synthesized using ßGBP from the crab Scylla serrata (Ss-ßGBP-ZnONP). Ss-ßGBP-ZnONP was observed as a 100 kDa band on sodium dodecyl sulfate polyacrylamide gel and characterized with UV-vis spectroscopy at 350 nm. X-ray diffraction analysis displayed values consistent with those for zincite. Fourier transform infrared spectroscopy revealed the presence of functional groups, including amide, alcohol, alkane, alkyl halide, and alkene groups. The zeta potential (-5.36 mV) of these particles indicated their stability, and transmission electron microscopy revealed the presence of 50 nm nanocones. Ss-ßGBP-ZnONPs were tested at 100 µg/mL against the gram-positive Enterococcus faecalis and gram-negative Pseudomanas aeruginosa using confocal laser scanning microscopy and the bacterial viability assay was also performed. The growth of MCF7 breast cancer cells was inhibited following treatment with 75 µg/mL Ss-ßGBP-ZnONPs. Thus, Ss-ßGBP-ZnONPs have the ability to control the growth of pathogenic bacteria and inhibit the viability of MCF7 breast cancer cell lines.

Anti-biofilm properties and immunological response of an immune molecule lectin isolated from shrimp Metapenaeus monoceros.

The study is carried out to understand the antimicrobial and immunological response of a potential immune molecule lectin, MmLec isolated from haemolymph of Speckled shrimp, Metapenaeus monoceros. MmLec was purified using mannose coupled Sepharose CL-4B affinity chromatography, which was further subjected on SDS-PAGE to ascertain the distribution of their molecular weight. Sugar binding specificity assay was conducted at various pH and temperatures to investigate the binding affinity of MmLec towards the specific carbohydrate molecule. Functional analysis of immune molecule MmLec included haemagglutination assays performed using human erythrocytes and yeast agglutination activity against Saccharomyces cerevisiae which, were analyzed using light microscopy. In order to study the antimicrobial activity, two Gram-negative (Vibrio parahaemolyticus and Aeromonas hydrophila) and two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria were treated with purified MmLec. Moreover, these bacterial species were also treated at different concentration of the MmLec to speculate the antibiofilm properties of MmLec which was analyzed under Light Microscopy and Confocal Laser Scanning Microscopy. In addition, other functional characterization of MmLec showed the uniqueness of MmLec in agglutination of human erythrocyte as well as the cells of yeast Saccharomyces cerevisiae. Also, the phenoloxidase activity and encapsulation assay was evaluated. MTT assay displayed that MmLec are potent in anticancer activity. The study will help to understand the immunological interference and antimicrobial nature of MmLec which would be supportive in establishing a potential therapeutic tool and to develop better and novel disease control strategies in shrimp and farmed aquaculture industries as well as in health management.

Antimicrobial properties and phenoloxidase activation of the lectin isolated from kadal shrimp (Metapenaeus dobsoni).

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study.

Antimicrobial and biochemical characterization of a C-type lectin isolated from pearl spot (Etroplus suratensis).

The present study reveals purification and characterization of a C-type lectin from the serum of pearl spot, Etroplus suratensis (Es-Lec). The Es-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 75 kDa in SDS-PAGE. The surface morphology of purified Es-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 2.958 min was appeared in high performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis expresses a single peak at 31.8372̊ and MALDI-TOF peaks which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Es-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy and haemagglutination inhibition was also done. In addition, purified Es-Lec showed the broad spectrum of antibacterial activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila. Antibiofilm potential of purified Es-Lec against selected Gram-negative bacteria exhibited the disruption of biofilm architecture at the concentration of 50 µg ml-1 and also it exhibited antiviral and anticancer activity.

Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillus subtilis A1.

In this work, the biodegradation of the crude oil by the potential biosurfactant producing Bacillus subtilis A1 was investigated. The isolate had the ability to synthesize degradative enzymes such as alkane hydroxylase and alcohol dehydrogenase at the time of biodegradation of hydrocarbon. The biosurfactant producing conditions were optimized as pH 7.0, temperature 40°C, 2% sucrose and 3% of yeast extract as best carbon and nitrogen sources for maximum production of biosurfactant (4.85 g l-1). Specifically, the low molecular weight compounds, i.e., C10-C14 were completely degraded, while C15-C19 were degraded up to 97% from the total hydrocarbon pools. Overall crude oil degradation efficiency of the strain A1 was about 87% within a short period of time (7 days). The accumulated biosurfactant from the biodegradation medium was characterized to be lipopeptide in nature. The strain A1 was found to be more robust than other reported biosurfactant producing bacteria in degradation efficiency of crude oil due to their enzyme production capability and therefore can be used to remove the hydrocarbon pollutants from contaminated environment.