Papers by Mukesh Pasupuleti
A B S T R A C T Superoxide dismutase (SOD) is an evolutionary conserved detoxification enzyme and... more A B S T R A C T Superoxide dismutase (SOD) is an evolutionary conserved detoxification enzyme and powerful antioxidant which defends against the elevated ROS that are induced by various stresses. Arthrospira platensis (Ap) is known for its antioxidant-mediated immunostimulant properties, but there is no report on the SOD dependent anti-oxidant mechanism. Therefore, in this study, we have analysed the effect of H 2 O 2 on growth and pigment composition in spirulina. Results showed that spirulina exposed to 10 mM H 2 O 2 showed elevated growth pattern as well as increase in chlorophyll pigment composition especially during early days of exposure. Gene expression results showed that the expression profile of ApSOD during oxidative stress stimulated by 10 mM H 2 O 2 at different time intervals (0, 5, 10, 15 and 20 days) with highest expression on day 10 post-exposure. Together, the results confirmed the antioxidant role of ApSOD in spirulina during oxidative stress induced by H 2 O 2. Based on the amino acid arrangement and composition, we have predicted a short peptide 160 LGLDVWEHAYYL 171 (LL12) from the catalytic centre of C-terminal SOD domain; further the peptide was synthesized. Antioxidant assays showed that LL12 peptide critically involved in radical scavenging mechanism. Also, LL12 peptide reduced the intracellular ROS level in H 2 O 2 exposed leucocytes at a concentration of 12.5 μM. Cytotoxicity assay was performed on human leucocytes which showed that LL12 did not exhibit any cytotoxic activity against any of the leucocytes population. Overall, the study highlights the radical scavenging property of a novel short peptide derived from the C-terminal domain of ApSOD which have the potential to develop as a biopharmaceutical drug.
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Fish & shellfish immunology, Jan 24, 2018
Channa striatus is one of the economically important freshwater fish with high demand in Southeas... more Channa striatus is one of the economically important freshwater fish with high demand in Southeast Asia for its nutritional and medicinal values. The unique composition of skin mucus of murrel provides immunity against pathogens; however, they are susceptible to few bacterial pathogens especially Aeromonas hydrophila. Although few immune molecules such as antimicrobial peptides have already been identified from the murrel mucus, there is no report on the complete gene profile of the skin and mucosal immunity. Therefore, in this study we applied transcriptome approach to identify the mRNA sequences of various immune molecules such as antimicrobial peptides, complement factors and adaptive immune molecules from the skin tissue. Transcriptome wide search revealed unique mRNA sequences of 13 antimicrobial peptides, 11 complement components, 2 major histocompatibility complex proteins and its receptor, 6 butyrophilins, 2 leptins and its receptor. Brief bioinformatics analysis of the iden...
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International journal of biological macromolecules, Jan 21, 2017
Chitinases play a vital role during the pathogenic invasion and immunosuppression in various orga... more 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 microsco...
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Developmental & Comparative Immunology, 2017
Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition ... more Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.
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Molecular Immunology, 2016
The antimicrobial peptides (AMPs) are multifunctional molecules which represent significant roles... more The antimicrobial peptides (AMPs) are multifunctional molecules which represent significant roles in the innate immune system. These molecules have been well known for decades because of their role as natural antibiotics in both invertebrates and vertebrates. The development of multiple drug resistance against conventional antibiotics brought a greater focus on AMPs in recent years. The cationic peptides, in particular, proven as host defense peptides and are considered as effectors of innate immunity. Among the various innate immune molecules, functions of pellino-1 (Peli-1) have been recently studied for its remarkable role in specific immune functions. In our study, we have identified Peli-1 from the cDNA library of freshwater prawn Macrobrachium rosenbergii (Mr) and analyzed its features using various in-silico methods. Real time PCR analysis showed an induced expression of MrPeli-1 during white spot syndrome virus (WSSV), bacteria (Vibrio harveyi) and lipopolysaccharide (LPS) from Escherichia coli challenge. Also, a cationic AMP named MrDN was derived from MrPeli-1 protein sequence and its activity was confirmed against various pathogenic bacteria. The mode of action of MrDN was determined to be its membrane permeabilization ability against Bacillus cereus ATCC 2106 as well as its DNA binding ability. Further, scanning electron microscopic (SEM) images showed the membrane disruption and leakage of cellular components of B. cereus cells induced by MrDN. The toxicity of MrDN against normal cells (HEK293 cells) was demonstrated by MTT and hemolysis assays. Overall, the results demonstrated the innate immune function of MrPeli-1 with a potential cationic AMP in prawn.
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ABSTRACT The striped murrel Channa striatus is one of the most commercially important freshwater ... more ABSTRACT The striped murrel Channa striatus is one of the most commercially important freshwater fish due to its high quality flesh, market demand and medicinal properties. However, in recent years, its inland culture practices have been severely affected due to various pathogenic infections. Currently an effective treatment for the infections is still lacking. We believe that an understanding of the immune system of this fish against biotic agents using molecular techniques would lead a possible way to control infections. Thioredoxin is a fish cytokine which play a role in innate immunity. Thioredoxins (TRxs) are a family of small, highly evolutionarily conserved immune regulatory cytokines which a crucial role in the cellular homeostasis. In this study, a cDNA encoding thioredoxin (TRx) was identified from the established C. striatus (named as CsTRx) cDNA library using GS-FLX™ technique. The nucleotide and protein sequence analysis revealed that the full length of CsTRx consists of 327 base pairs nucleotides with a 324bp open reading frame (ORF), which encodes for 108 amino acids long protein. Further analysis showed that the CsTRx polypeptide contains a thioredoxin domain between Val2 and Asn106. Multiple sequence alignments revealed that a CGPC motif and a hydrophobic dimerization interface were highly conserved in all organisms taken for analysis. The CGPC motif is responsible to control protein function via the redox state of structural or catalytic thiol group (Nkabyo et al. 2002). Phylogenetic study showed that CsTRx clustered together with vertebrate TRx1. Based on the analysis, it is confirmed that CsTRx belongs to TRx1s family. The secondary structure of CsTRx protein contained 44% α-helixes and 18% β-sheets. The tertiary structure of CsTRx fold showed 4 α-helixes and 4 β-sheets connected with random coils. Based on the computational biological analysis of CsTRx, we focused further to demonstrate CsTRx as a novel antioxidant agent.
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ABSTRACT Fishes are intermediate to species possessing innate immunity (invertebrates) and to org... more ABSTRACT Fishes are intermediate to species possessing innate immunity (invertebrates) and to organisms depending more on adaptive immunity (mammals). Chemokines are a super family of cytokines (8~10 kDa), acting as key regulator in immune response and in bridging innate and adaptive immunity. In this study, a full length CC chemokine 1 CDNA from striped murrel Channa striatus (CsCC1) has been using various bioinformatics tools. The analysis revealed that, CsCC1 is 641 base pairs (bp) in length, with a 5’ untranslated region (UTR) of 87 bp, a open reading frame (ORF) of 438 bp and 3’ UTR of 116 bp. The predicted CsCC1 polypeptide contains 146 amino acids, it has an iso-electric point of 9.3 and a theoretical molecular weight of 15kDa. Preliminary observations on motif and domain analysis showed that, CsCC1 possessed a single stretch of serine rich region at 93-133. Further motif analysis indicated that CsCC1 contains few high probability common motifs including four Protein Kinase C phosphorylation site, three Casein Kinase II phosphorylation site and one N- myristoylation site. Further investigation revealed that, CsCC1 also possesses a signal peptide region between 1and 26 and a small cytokine region between 30 and 88. The typical CC motif of CC chemokines is present at C33-C34. Multiple sequence alignment of CsCC1 with other known CC chemokines from teleost fishes showed showed more conserved regions than the other vertebrate considered for analysis. CC chemokines have been grouped into inducible and constitutive proteins, on the basis of their expression pattern. Phylogenetic tree was constructed from the homology data, considering Homo sapiens as the reference point. The tree revealed that, CsCC1 along with CsCC20 and CsCC19 of Channa striatus aligned with chemokines belonging to constitutive class. The 3D structural analysis of CsCC1 showed five helices (30.1%), two ß strands (6.84) and random coils (63.01). This study provides important information for further investigation of CsCC1 involvement in innate immunity.
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J Biol Chem, 2006
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ABSTRACT Freshwater prawn Macrobrachium rosenbergii is highly susceptible to various pathogens wh... more ABSTRACT Freshwater prawn Macrobrachium rosenbergii is highly susceptible to various pathogens which increase its mortality drastically. The prophenoloxidase (ProPO) activation system in prawn plays an important role in defense against pathogens and parasites. The ProPO system is activated by a group of enzymes (Lee et al. 2002), among them serine protease (SP) is an important protease that activates the ProPO system. To understand the role of SP in the mechanism of ProPO activating system, we identified a SP like protein from an assembled M. rosenbergii (named as MrSP) cDNA library by GS-FLX™ sequencing technique and studied its bioinformatics characteristic features. The full length MrSP cDNA has 1739 bases with an open reading frame of 1668 bases. The deduced protein contains 556 amino acids with a predicted molecular weight of 61 kDa and an isoelectric point of 5.3. Local alignment search revealed that MrSP is similar to masquerade like serine protease of Penaeus monodon (63%). Motif and pattern analysis showed that MrSP contains a trypsin like domain at 290-538 along with a modified catalytic triad His341-Asp391-Gly488 (Gly488 replacing serine) and this Gly488 inhibits the proteolytic activity of catalytic triad. Similarly, in the substrate binding site (Asp486-Ala514-Gly516) also serine is replaced by Ala514. The deduced amino acid of MrSP contains 6 conserved cysteine residues and making 3 disulphide bonds at C424-C498; C457-C478 and C488-C519. Phylogenetic analysis categorizes MrSP as a prophenoloxidase activating serine protease, since it is grouped with such proteases. Based on the bioinformatics characteristic as well as structural analysis, it is tempting to suggest that the characterized protein MrSP is a ProPO activating serine protease like protein. Moreover, the analysis showed that MrSP is involved in the prophenoloxidase activation system. Hence we extended our further investigation to study the involvement of MrSP in the ProPO activating enzyme cascade.
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Antimicrobial Agents and Chemotherapy, 2009
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Http Dx Doi Org 10 1080 10826068 2013 797434, Nov 25, 2013
Phosphate released from phytic acid can be used as a measure of phytase activity. However, most o... more Phosphate released from phytic acid can be used as a measure of phytase activity. However, most of the phosphate estimation methods have not examined the interference or interaction of phytic acid in the assay. In this article, we report the kinetics and influence of unreduced phytic acid on phosphate estimation by three of the often-used methods for phytase estimation, the AOAC, Cooper-Gowing, and Fiske-Subbarow methods. Our results show that the AOAC method is most suitable to estimate the phytase activity in the presence of phytate in the medium. In the Fiske and Subbarow method, we noticed that the time factor plays a role in the interference of the phytic acid; especially the readings taken during the second hour of incubation are influenced by the presence of phytic acid. The method of Cooper and Gowing is labor-intensive and is prone to give error values at higher concentrations.
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Molecular Immunology, 2015
In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial... more 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.
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Gene, Jan 21, 2015
Cu/ZnSOD (copper/zinc superoxide dismutase) primarily scavenges cytosolic reactive oxygen species... more Cu/ZnSOD (copper/zinc superoxide dismutase) primarily scavenges cytosolic reactive oxygen species (ROS) by converting ROS to hydrogen peroxide, which is then converted to water by the catalytic action of catalase, thus playing a pivotal role in the first line of defense mechanism against oxidative stress. In this study, we have reported a complete molecular characterization of cDNA sequence from striped murrel Channa striatus (Cs). Cellular location prediction reveals that CsCu/ZnSOD protein is cytosolic with an accuracy of 90%. Phylogenetic analysis showed that CsCu/ZnSOD belongs to SOD1 group and it shared a common clad with Asian seabass Lates calcarifer and then with other fishes. The highest CsCu/ZnSOD gene expression, SOD enzyme activity and total protein concentration were observed in the liver and its regulation was studied upon fungus (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) challenges. Based on the results obtained from the above analysis, we concluded a...
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Innate immunity, 2013
Innate immunity is triggered by a variety of bacterial molecules, resulting in both protective an... more Innate immunity is triggered by a variety of bacterial molecules, resulting in both protective and potentially harmful pro-inflammatory responses. Further, innate immunity also provides a mechanism for the maintenance of homeostasis between the host immune system and symbiotic or non-pathogenic microorganisms. However, the bacterial factors that mediate these protective effects have been incompletely defined. Here, it was demonstrated that the lantiobiotic nisin Z is able to modulate host immune responses and mediate protective host immunity. Nisin Z induced the secretion of the chemokines MCP-1, IL-8 and Gro-α, and significantly reduced TNF-α induction in response to bacterial LPS in human PBMC. The results correlated with the ability of nisin Z to confer protection against both the Gram-positive organism Staphylococcus aureus, and the Gram-negatives Salmonella enterica sv. Typhimurium and Escherichia coli in murine challenge models. Mechanistic studies revealed that nisin Z modula...
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Freshwater prawn Macrobrachium rosenbergii is one of the commercially important food species of S... more Freshwater prawn Macrobrachium rosenbergii is one of the commercially important food species of South Asia. However, viral and bacterial infectious diseases have a serious impact on this species. Prawn defends against these pathogens through innate immunity. One of the most important components of innate immunity is antimicrobial peptide (AMPs). Histones are a family of positively charged proteins, highly conserved in eukaryotic cells. The antimicrobial activity of histones and their derived products have been shown in many organisms including invertebrates and vertebrates. Thus, molecular characterization of antimicrobial peptides would supply a thorough knowledge about the immune system of prawn and provide a new understanding on disease control strategy. In order to characterize a histone from M. rosenbergii (designated as MrHis) at molecular level, a full length cDNA of MrHis was identified from the established M. rosenbergii cDNA library using Genome Sequencing FLX™ (GS-FLX™) t...
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Fish Physiology and Biochemistry, 2014
We have reported the molecular characterization including gene silencing, superoxide activity, su... more 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 β-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.
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The purpose of this study was to determine the distribution of virulent genes, stx1, stx2 and hly... more The purpose of this study was to determine the distribution of virulent genes, stx1, stx2 and hlyA as well as antibiotic profiling among Shiga-Toxigenic E. coli isolatedfrom food samples. One hundred and thirty four strains of E. coli isolated from 300 food samples were subjected to PCR analysis. Antibiotic sensitivity profiling was also performed. Of the 31 STEC positive strains, 25.8% exhibited only stx1 gene, 54.8% exhibited both stx1 and stx2 genes, 9.6% exhibited stx1+ hlyA genes, 3.2% exhibited stx2+hlyA genes and 7.1% isolates were positive for all the three genes stx1+stx2+hlyA studied. About 32.3% of STEC elicited eight different antibioticresistance patterns ranging from one to eight widely used antibiotics. There was an overall uneven distribution of all the three virulence genes in the STEC strains. The stx1 gene and the stx1+stx2 combination were the most prevalent in the STEC strains. This study indicates that the bacteria which contain both types of shiga toxin were resistant to most of the antibiotics tested. Efficient cooking and other good hygienic prophylactic measures are needed to decrease the incidence of STEC in food items.
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Papers by Mukesh Pasupuleti