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Prolyl oligopeptidase (POP) plays a crucial role in the processing and degradation of neuropeptides and regulates inositol trisphosphate (IP3) signaling in mammals. We have reported that POP inhibition leads to IP3-mediated calcium efflux leading to mitochondrial-mediated apoptosis in the filarial parasite Setaria cervi. This study further elucidates the effect of altered calcium homeostasis on the proteome of filarial parasites. Adult parasites were treated with POP's specific inhibitor, Z-Pro-prolinal (ZPP), for 7 h. Cytosolic and mitochondrial proteome was analyzed using 2D gel electrophoresis coupled with MALDI-MS/MS. Phosphoproteins were also analyzed in the cytosolic fraction of the parasites. The phosphoprotein analysis revealed 7, and 9 spots in the cytosolic fraction of control and ZPP-treated parasites, respectively. The two identified protein spots in the treated set were found to be involved in G protein signaling. In cytosolic fraction, 109 and 112 protein spots were observed in control and treated parasites, respectively. Of these, 56 upregulated and 32 downregulated protein spots were observed in the treated set. On the other hand, 50 and 47 protein spots were detected in the mitochondrial fraction of control and treated parasites, respectively. Of these spots, 18 upregulated and 12 down-regulated protein spots were found in treated parasites. In silico analysis showed that the identified proteins were involved in energy metabolism, calcium signaling, stress response, and cytoskeleton organization. These findings correlate with our previous results suggesting the important regulatory role of POP in signaling and different metabolic pathways of filarial parasites.

The available antifilarial medications are effective only against the larval stage of the filarial parasite. As a result, there is a pressing need for an adulticidal drug. The development of drugs requires the identification of molecular targets that are critical for parasite life. In this study, we observed the effect of 17-N-allyl-17-demethoxygeldanamycin on the survival of adult filarial parasites. The 17-N-allyl-17-demethoxygeldanamycin (17-AAG) is a derivative of geldanamycin (GA), which is an inhibitor of heat shock protein (HSP)90. It is less toxic as compared to geldanamycin. The motility and viability of the adult filarial parasite Setaria cervi were decreased on exposure to 17-AAG at 2.5 and 5.0 μM/ml concentrations. The 17-AAG treated parasites showed induction of oxidative stress as evidenced by decreased activity of various antioxidant enzymes like glutathione s-transferase, glutathione reductase, thioredoxin reductase, and an increase in ROS production in comparison to control. Oxidative stress may lead to altered calcium homeostasis. Indeed, in 17-AAG treated worms, there was a rise in calcium in the cytosol and mitochondria, as well as a decrease in the ER. We also observed enhanced activity of phospholipase C in the treated parasite, suggesting the opening of calcium channels located on the ER membrane. ER stress is marked by a reduced level of protein disulfide isomerase. Further, 17-AAG treated worms showed an increase in apoptotic marker enzyme activities like calpain, cyt-c, and caspase-3. The 2D-gel electrophoresis technique showed 142 protein spots in the control and 112 spots in the 17-AAG treated parasite. Thus, 17-AAG induced oxidative stress, and altered calcium, and proteostasis of parasites, which led to apoptosis.

Lymphatic filariasis caused by filarial nematode is an important disease leading to considerable morbidity throughout tropical countries. Even after specific elimination programs, the disease continue to spread in endemic countries. Thus newer therapeutic interventions are urgently needed to control the spread. In the present study, we have seen the effect of andrographolide (andro), a diterpenoid lactone from the leaves of Andrographis paniculata on filarial parasite Setaria cervi. There was time and concentration dependent decrease in motility and viability leading to death of parasite after 6 h of the exposure of andro. Andro showed potential antifilarial activity with an IC

GRP94, a member of HSP90 family, is involved in folding and degradation of endoplasmic reticulum proteins. The proteome analysis of Setaria cervi, a bovine filarial parasite showed that a 91 kDa protein was over expressed, after the parasites were maintained in glucose deprived medium. The MALDI- LC/MS analysis of the 91 kDa band confirmed it as endoplasmin precursor (GRP94). Amino acid sequence alignment of S.cervi GRP94 exhibited maximum similarity with human filarial parasite Wuchereria bancrofti, Brugia malayi and Loa loa GRP94. Tunicamycin treatment of S. cervi worms revealed that the expression of GRP94 is associated with ER stress. Transcription of S. cervi grp94 as well as igf is regulated by transcription factors ATF-6 and XBP-1S which was confirmed by Real Time PCR. Moreover, marked alteration in the expression of igf after 3 h and 6 h of drug treatment suggested propagation of survival pathway under ER stress. The activities of ER stress markers protein disulphide isomerase and glycosyltransferase were significantly reduced after 6 h of tunicamycin treatment. The present findings thus indicate that the expression of GRP94 and regulation of its expression is under ER stress in Setaria cervi. To our knowledge this is the first report of identification of GRP94, in any filarial parasite till date.

AIM Identification of a 29 kDa heat stress protein in filarial parasite Setaria cervi and evaluation of its diagnostic potential against lymphatic filariasis. METHODS AND RESULTS The Heat shock proteins (HSPs) were induced in filarial parasite S cervi by incubated at 42°C for 2 hours. The 10% SDS-PAGE of cytosolic extract showed several over-expressed bands. The MALDI-LC/MS analysis of 29 kDa band showed 100% similarity with Bm14-3-3 like protein 2. Multiple sequence alignment of Bm14-3-3 like protein 2 sequence with W bancrofti, Caenorhabditis elegans; Loa loa and Homo sapiens showed 100%, 86%, 83% and 78%, sequence similarity respectively. The antigenic efficacy of Sc14-3-3 protein was evaluated with different filarial sera using ELISA which showed cross-reactivity in order to Endemic Normal (EN)

A 75 kDa serine protease having prolyl oligopeptidase activity has been purified from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 6 peptides showing nearest match S9A (prolyl oligopeptidase) family protein from Plesiocystis pacifica. The ScPOP was found to be unique compared to mammalian POP with respect to its kinetic properties. To elucidate its role, filarial parasites were exposed to specific inhibitor of POP, Z-Pro-prolinal (ZPP) for 8 h. The inhibition of POP induced calcium signaling via phospholipase c stimulation which further triggered mitochondrial mediated apoptosis in filarial parasites.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 μM. Albendazole also inhibited PGK competitively with Ki value 35.39 μM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.

Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01–5.0 μM) and calcium chloride (0.01–100 μM). The rBmPGK was activated up to 85% in the presence of CaM at 1 μM and 10 μM concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ± 10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0 μM and 1.0 mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain. On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

In this ex vivo study, S. cervi parasitoses were treated with Ivermectin (50 μM), Albendazole (200 μM) alone and Ivermectin + Albendazole (50 + 200 μM) at 37°C for 8 h and the motility and viability of the parasitoses were evaluated. Individually both drugs Ivermectin (Iver) and Albendazole (Alb) are reported to affect the function and integrity of ER, however till date, no reports are available on the functional changes in ER due to a combined Iver and Alb treatment of bovine helminth parasitosis. Here, we report the lethal effect of a combination treatment of Iver and Alb against adult bovine filarial parasitosis Setaria cervi. The underlying mechanism of drug action was elucidated by performing a systematic biochemical, molecular and proteomics based study. Altered calcium homeostasis in drug treated parasitoses lead to reduction in levels of total Endoplasmic Reticulum (ER) calcium by 50 % and 61 % and elevation by 50 % and 63 % in cytosol in Iver alone and Iver + Alb treated parasitoses respectively. Further, it was found that upregulated expression of ER localized GRP94, galactosyltransferase and glycosyltransferase activity in addition to reduction in activity of PDI indicated ER stress mechanisms being operative under combined drug treatment. Marked rise of 79 % reactive oxygen species and reduced antioxidant levels induced oxidative stress in drug treated parasitosis. The collective effect of both ER and oxidative stress might have triggered apoptosis, as evidenced by the elevated calpain activity, reduction of 67 % in cytochrome c oxidase and 83 % rise in caspase-3 activity in the Iver + Alb treated parasitoses respectively. The ER proteome analysis by 2D gel electrophoresis revealed 76 spots in the control and 56 spots in the treated proteome. A MALDI-MS/MS analysis of some of the differentially expressed spots of the combination drug treated parasitoses identified glucuronosyltransferase as a major upregulated protein with a fold change of 1.81. Trafficking protein, acyl transferase, MATH involved in protein folding were also found to be downregulated. Thus, this study based on biochemical and proteomic approaches indicates that a combination of anti-filarial drugs Iver and Alb can alter calcium homeostasis in bovine filarial parasitosis leading to induction of ER stress culminating into apoptosis.

The arsenic tolerant bacterial strains Staphylococcus arlettae (NBRIEAG-6), Staphylococcus sp. (NBRIEAG-8) and Brevibacillus sp. (NBRIEAG-9) were tested for their roles in enhancing plant growth and induction of stress-related enzymes in rice (Oryza sativa L. cv. NDR-359) plants at two different concentrations, 30 and 15mg/kg of As(V) and As(III), respectively. An experiment was conducted to test the effect of these strains on plant growth promotion and arsenic uptake. We found 30%-40% reduction in total As uptake in bacteria-inoculated plants, with increased plant growth parameters compared to non-inoculated plants. Moreover, the bacteria-inoculated plants showed reduced activity of total glutathione (GSH) and glutathione reductase (GR) compared to their respective controls, which suggests the bacteria-mediated reduction of oxidative stress in plants. Thus, these strains were found to be beneficial in terms of the biochemical and physiological status of the plants under arsenic stress conditions. Furthermore, one-way ANOVA and principal component analysis (PCA) on enzymatic and non-enzymatic assays also revealed clear variations. The results support the distinction between control and treatments in both shoots and roots. Therefore, this study demonstrates the potential of rhizobacteria in alleviating arsenic stress in rice plants.

Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.

Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis.

Arsenic (As), a toxic metalloid adversely affects plant growth in polluted areas. In the present study, we investigated the possibility of improving phytostablization of arsenic through application of new isolated strain Brevundimonas diminuta (NBRI012) in rice plant [Oryza sativa (L.) Var. Sarju 52] at two different concentrations [10ppm (low toxic) and 50ppm (high toxic)] of As. The plant growth promoting traits of bacterial strains revealed the inherent ability of siderophores, phosphate solubilisation, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production which may be associated with increased biomass, chlorophyll and MDA content of rice and thereby promoting plant growth. The study also revealed the As accumulation property of NBRI012 strain which could play an important role in As removal from contaminated soil. Furthermore, NBRI012 inoculation significantly restored the hampered root epidermal and cortical cell growth of rice plant and root hair elimination. Altogether our study highlights the multifarious role of B. diminuta in mediating stress tolerance and modulating translocation of As in edible part of rice plant.

The currently available antifilarial drugs are mostly effective against microfilariae. Antifilarial drugs have disadvantages such as toxicity and the development of resistance due to the continuous use. Therefore alternative drugs are required for the control of disease. At present, nanoparticles are used for developing anti-parasitic therapy for their special properties such as smallest in size, bio-availability, bio-compatibility and penetration capacity into a cell. In the present study green nanoparticles were biosynthesized by using leaf extract of Andrographis paniculata to evaluate its antifilarial efficacy. Biosynthesized nanoparticles were characterized by UV–Vis spectroscopy, FTIR, XRD, SEM, EDAX, TEM, HRTEM, AFM and found to be homogenous with average size of 11 nm. The antifilarial efficacy of green nanoparticles was based on adult filarial parasite motility and viability assay. These green nanoparticles (NPs) were found to have antifilarial activity with LC50 of 11.6 μg/ml against adult female filarial parasites. The green nanoparticles induced oxidative stress as evidenced by elevated ROS production and decline of parasitic GST, GR, TRxR and GSH levels in the parasite. The activation of ced-3 gene, a homolog of mammalian caspase 3, reduced expression of ced-9 and decreased activity of cytochrome c oxidase suggested induction of mitochondrial mediated apoptosis in parasite. These green NPs are more effective than the plant extract. This is the first report where green nanoparticles synthesized from A. paniculata showed antifilarial efficacy against adult filarial parasites.

Thioredoxin reductase plays a crucial role in the maintenance of cellular redox homeostasis. In this study, we have targeted TrxR in Setaria cervi , a bovine filarial parasite using its inhibitor CDNB. It caused significant decrease in the motility and viability of these parasites leading to their death. Inhibition of TrxR leads to the downregulation of the antioxidant system followed by generation of oxidative stress in these parasites. The increased ROS level induced lipid peroxidation and protein carbonyl formation which might alter the mitochondrial membrane permeability leading to release of cytochrome c. CDNB significantly downregulated the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also upregulated ced-3, homolog of mammalian caspase 3 suggesting initiation of intrinsic pathway of apoptosis. The proteomic profile of CDNB treated parasites showed marked alteration in abundance of different protein spots with 20% downregulated and 13% unregulated spots in comparison to control parasites. We observed a downregulation in the glycolytic enzymes such as enolase, PGK, and GAPDH thereby blocking the ATP formation in the parasite. This study suggests that TrxR inhibition disrupts the cellular homeostasis thereby generating oxidative stress followed by mitochondrial mediated apoptosis in filarial parasites leading to the death of the parasites. Biological significance Lymphatic filariasis is one of the most prevalent tropical diseases caused by tissue dwelling parasitic nematodes viz., Wuchereria bancrofti , Brugia malayi and Brugia timori . Currently available antifilarial drugs effectively eliminate larval stages of the parasite but are ineffective against the adult worms. Therefore, there is an urgent need for finding proteins/enzymes which play a crucial role in the persistence of these parasites. Our study for the first time reports the important role played by S. cervi TrxR in its survival. Thus, suggesting filarial TrxR as a potent chemotherapeutic target against lymphatic filariasis. This would help in screening of new compounds having macrofilaricidal activity.

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of deoxythymidine triphosphate, which is an essential component for DNA synthesis. The gene encoding thymidylate kinase of Brugia malayi was amplified by PCR and expressed in Escherichia coli. The native molecular weight of recombinant B. malayi thymidylate kinase (rBmTMK) was estimated to be ∼52 kDa by gel filtration chromatography, suggesting a homodimeric structure. rBmTMK activity required divalent cation and Mg2+ was found to be the most effective cation. The enzyme was sensitive to pH and temperature, it showed maximum activity at pH 7.4 and 37 °C. The Km values for dTMP and ATP were 17 and 66 μM, respectively. The turnover number kcat was found to be 38.09 s−1, a value indicating the higher catalytic efficiency of the filarial enzyme. The nucleoside analogues 5-bromo-2′-deoxyuridine (5-BrdU), 5-chloro-2′-deoxyuridine (5-CldU) and 3′-azido-3′-deoxythymidine (AZT) showed specific inhibitory effect on the enzyme activity and these effects were in good association with binding interactions and the scoring functions as compared to human TMK. Differences in kinetic properties and structural differences in the substrate binding site of BmTMK model with respect to human TMK can serve as basis for designing specific inhibitors against parasitic enzyme.

Summary More than 1·5 billion people are at risk of being infected with filarial nematodes worldwide. Therapy and control of transmission are mainly based on mass drug distribution. As these drugs have to be administered annually or biannually and might be loosing their efficacy, a vaccine against filariae is an alternative approach to chemotherapy. In the current study, we have analysed the potential of Brugia malayi heat shock protein 70 (BmHsp70) as a vaccine candidate in a murine helminth infection. Immunization of BALB/c mice with alum-precipitated recombinant BmHsp70 conferred partial protection against subsequent challenge infection with the rodent parasite Litomosoides sigmodontis. Immunization resulted in reduced numbers of larvae in the pleural cavity as well as reduced numbers of circulating microfilariae. Reduced parasite burden was associated with high titres of BmHsp70-specific antibodies and increased production of type I and II cytokines in response to L. sigmodontis antigen and BmHsp70. In summary, the immunization with BmHsp70 induced cellular and humoral immune responses and partially protected against L. sigmodontis in a challenge infection. Therefore, we hypothesize that BmHsp70 might be considered as a potential vaccine candidate for reduction in the incidence of B. malayi infections in future studies.

A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP.

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l−1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l−1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

The present study reports the arsenic (As) tolerance mechanism of bacteria Bacillus aryabhattai (NBRI014). The data explores the intracellular accumulation and volatilization of As from the culture medium after 48 h of exposure to 25,000 mg l −1 arsenate As(V). The study also provides the evidence of presence of ars operon in bacteria, which may have played an important role in reducing As toxicity. Additionally, we found 7 differentially expressed proteins to be up-regulated in bacterial cells upon As exposure which may have role in reducing As toxicity inside bacterial cells. Furthermore, Fourier transform infrared (FTIR) spectroscopic techniques were useful to describe the structural and compositional alterations in bacterial cells after As treatment. It showed the changes in peak positions of the spectrum pattern when NBRI014 was grown in medium containing As, indicating that these functional groups viz. (amino, alkyl halides and hydroxyl) present on bacterial surface, which may be involved in As binding. The above results signify that biotechnological application of the isolate NBRI014 could be helpful in removal of As from polluted sites.

For survival, parasite exerts several lines of defense of which drug neutralization is one of the major phenomena. Lack of phase I cytochrome P450 in some of the nematode render them depend on the phase II detoxification system involving GST as a major detoxifying enzymes. In present study, the antifilarial DEC, phenolic compound BHA and methyl chalcone have been evaluated for proteomic and biochemical studies in Setaria cervi. BHA and methyl chalcone showed cytotoxic effect leading to irreversible inhibition in motility and viability of parasites. These drugs showed marked alteration in proteomic profile of S. cervi at 100 μM concentration with 10.82, 8.52 and 6.75% downregulated (0.5) and 7.64, 31.78 and 24.32% upregulated (1.5) in DEC, BHA and methyl chalcone treatment respectively. Significant depletion in GSH level with increase in NO production was observed. Amongst these compounds, methyl chalcone demonstrated significant inhibitory effect (p0.05) on GST, PGHS and PTP activity leading to loss of metabolic homeostasis and parasite death. The cytotoxic response and altered expression profile of major enzymes under drug exposure suggested the oxidative stress induced apoptosis as a major cause of parasite killing which was further supported by DNA fragmentation in BHA and methyl chalcone.

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 microM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.

Since glutathione-S-transferase (GST) mediated xenobiotic detoxification is a crucial mechanism in nematodes survival, we aimed to conduct an in silico analysis of filarial GST in order to predict the possible interactions for antifilarials. Present report depicts the homology modeling approach applied in the construction of molecular structure of Brugia malayi GST (BmGST) followed by its docking simulation with available antifilarials such as diethylcarbamazine, albendazole, Butylated Hydroxyanisole (BHA) and substituted chalcones. A very low root mean square deviation (0.82A) from template structure and stereochemical quality of constructed BmGST model proposed it as a significant framework for further analysis. In docking studies antifilarials and chalcones exhibited demarcation in their binding affinity and modes. Amongst all the compounds studied, albendazole and methyl-substituted chalcone showed the lowest binding energy and occupied binding pocket near to substrate binding site of GST. The side chain of these compounds interplayed as a potential interaction site which targeted mainly hydrophilic residues of the BmGST. The structural information and binding site mapping of BmGST for different antifilarials obtained from this study could aid in screening and designing new antifilarials or selective inhibitors for chemotherapy against filariasis.

Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory-secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH 5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH 3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host-parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.

Using synthetic peptide substrate Leu- p -NA, leucine aminopeptidase (LAP) activity was detected in both microfilarial and adult stages of a bovine filarial parasite Setaria cervi . A single protein fraction containing LAP activity was purified from the adult female S. cervi using three different chromatographic techniques. This purified enzyme was shown to be a 321 kDa zinc dependent metalloexopeptidase having maximum activity at pH 9.0 and 37 °C. Its activity was significantly inhibited by aminopeptidase specific inhibitors such as 1,10-phenanthroline, ethylene diaminetetraacetic acid (EDTA), amastatin and bestatin; and activated by Co 2+ , Mn 2+ and Mg 2+ ions. Puromycin and l -amino acids (e.g., glutamine, leucine and glycine) also showed some moderate inhibitory effects on the purified enzyme. Among various synthetic substrates tested, the purified enzyme hydrolysed Leu- p -NA at very high rate suggesting it to be a LAP. Both ELISA and western blotting analyses of S. cervi LAP revealed the presence of homologous protein in human filarial parasite Wuchereria bancrofti . The higher sensitivity of S. cervi LAP with microfilariaemic sera compared to other categories of W. bancrofti infected human sera implied its potential as a serodiagnostic marker against active filarial infection. The antigenic similarity between S. cervi LAP and W. bancrofti makes this molecule ideal for the discovery of new diagnostic marker, drugs and/or vaccine candidate for human lymphatic filariasis.

A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis.

In our continuing search for safe and efficacious antifilarials, a series of novel chalcone-benzothiazole hybrids have been synthesized and evaluated for their Brugia malayi thymidylate kinase (BmTMK) enzyme inhibition activity. Their selectivity towards BmTMK was studied and compared to the human TMK (HsTMK) by an in silico method. Out of seventeen derivatives, compounds 34 and 42 showed higher interactions with the BmTMK active site. MolDock docking model revealed the interactions of these two derivatives and the results corroborated well with their in vitro antifilarial activities. Our studies suggest that these hybrids are selective towards the BmTMK enzyme and may serve as potential therapeutic agents against filariasis.

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adultSetaria cervifemales using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity againstBrugia malayi(a human filarial parasite) in jirds (Meriones unguiculatus)in vitroandin situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells toB. malayimicrofilariae (mf) and infective larvae (L3)in vitroand induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi175 kDa antigen serum was more effective in inducing cytotoxicity toB. malayiL3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.

In vitroreleased products of adultSetaria cervifemales, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein releasedin vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectivly. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such asN-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition underin vitroconditions. Both enzymes were significantly inhibited by antibody IgG separated fromWuchereria bancroftiinfected human sera, showing a possible immunoprotective role.

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 μM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 μM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.

The age- and sex-specific distributions of human infections with Wuchereria bancrofti were investigated at two sites in the Varanasi region of north India: one a rural, agricultural area (Chiraigaon) and the other an urban-slum area (Sunderpur). A random clinical and parasitological survey revealed that the prevalence of microfilaraemia and elephantiasis in the urban area (14% and 7.3%, respectively) were both higher than in the rural area (9% and 3.1%, respectively). In both areas, prevalence of microfilaraemia generally increased with age, to a maximum in those aged 20-29 years, and then declined. Within most age-groups, the prevalences of microfilaraemia and elephantiasis were higher in males than females. However, the prevalence of microfilaraemia in females from Chiraigaon who were aged30 years was higher than in their male counterparts. Though individual microfilarial intensities varied greatly, the geometric mean microfilarial intensity was higher in Sunderpur than in Chiraigaon (214 v. 196 microfilariae/ml). All 83 subjects with elephantiasis, except one in Sunderpur, were amicrofilaraemic. The present results indicate that bancroftian filariasis is one of the major public-health problems in the study area.

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo . TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5′-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi ( S. cervi ). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi . Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi . The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643 U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~ 50 kDa while the optimum pH and temperature were found to be 7.0 and 35 °C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29 ± 1.40 kcal/mol. The Km and Vmax were determined to be 0.27 ± 0.045 mM; 30.30 ± 1.30 U/ml with NADPH and 0.59 ± 0.060 mM; 4.16 ± 0.095 U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1 μM concentration. The inhibition of ScGR activity with NAI (IC 50 0.71 mM), NEM (IC 50 0.50 mM) and DEPC (IC 50 0.27 mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.

In our earlier report, a 26 kDa Setaria cervi glutathione-S-transferase showed significant protection (82%) in jirds infected with L3 larvae of Brugia malayi. In the present study we have identified the major antigenic epitopes in ScGST. Carboxypeptidase B has been used to digest the ScGST in to smaller fragments. The digested products were separated as four protein bands on SDS-PAGE. The smallest fragment of 6 kDa (P4) from ScGST was identified as major antigenic epitope because of its significant reactivity with jird anti ScGST sera and human filarial sera in immunoblotting. The MALDI-LC/MS sequencing of ScGST P4 peptide (5KLTYFSIRGRGLAEPIRL20, 22KVPDDQQFLDDLISR36 and 47VFHFGQGPHHGPPR62) suggested that this protein band has a fragment of 5–62 residues long that matched with the N-terminal end of filarial GST. The antigenicity plot of ScGST was compared with BmGST model and both exhibited three immunogenic peaks within the first 60 residues towards N-terminal. In BmGST the N-terminal region was also detected with N-glycosylation signal peptide NAS adding to its high immunogenic property. Further, P4 showed strong reactivity with IgG1 and IL-4 response in endemic normal sera suggested its role in Th2 response which in turn is correlated with antibody dependent cell mediated cytotoxicity. Thus taking these results into account we propose 5–62 residues long N-terminal peptide of GST as a potential target for further vaccination studies against filarial infection.

SUMMARYSetaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16·66 mM, 25·0 μM/ml/min with pNPP; 20·0 mM, 40·0 μM/ml/min with phospho-L-tyrosine and 27·0 mM, 25·0 μM/ml/min with phospho-L-serine. KIwith pNPP and sodium orthovanadate (IC5033·0 μM) was calculated to be 50·0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity withW. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Underin vitroconditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest thatS. cerviDSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

Summary Lymphatic filariasis (LF) caused by Wuchereria bancrofti is widely prevalent in tropical and subtropical countries. Night blood film examination is most commonly used for diagnosis of filariasis but is cumbersome and labour intensive. In order to develop an indirect ELISA-based immunodiagnostic test, the importance of antifilarial IgG subclasses was evaluated in bancroftian filariasis patients. Blood samples from healthy individuals and different categories of LF patients were used to estimate the diagnostic potential of selenium glutathione peroxidase antigen purified from the bovine filarial parasite Setaria cervi . This antigen reacted with both IgG 1 and IgG 4 ; however, the IgG1 response was greater in microfilaraemic patients and the IgG 4 response was higher in chronic filarial patients. The diagnostic sensitivity of IgG 1 and IgG 4 was 97% and 96% whereas specificity was determined to be 95% and 98% respectively. Our observations suggest that SeGSHPx could be an alternative diagnostic marker for the detection of bancroftian filariasis in an endemic area.

Experimental evidence has shown that Setaria cervi a bovine filarial parasite contains significant amount of prostaglandin H synthase like activity in the somatic extract of its different life stages. A protein with characteristics of prostaglandin H synthase was purified to homogeneity from female somatic extract using a combination of affinity and gel filtration chromatography. Molecular weight of purified enzyme was 70 kDa as determined by SDS-PAGE. Purified enzyme showed high activity with arachidonic acid and TMPD substrates suggests the presence of both cyclooxygenase and peroxidase activity in enzyme. Fluorescence spectroscopy and hemin-associated peroxidase activity confirmed presence of heme in purified enzyme. The Km and Vmax values using arachidonic acid were determined to be 79 ± 1.5 μM and 0.165 ± 0.2 U/ml, respectively. Further, indomethacin and aspirin, specific inhibitors for PGHS, significantly inhibited the enzyme activity. Diethylcarbamazine, an antifilarial drug inhibited the microfilarial PGHS like activity as well as their motility. Here we are reporting for the first time PGHS like activity in filarial parasite and its inhibition with DEC which provide that this enzyme could be used as a drug target.

Using mass spectrometry and immunological approaches, a heat shock protein 70 associated with lymphatic filariasis (LF) has been identified from a bovine filarial parasite Setaria cervi. A heat shock protein was detected in different life stages of S. cervi when exposed to an elevated temperature of 44 degrees C. A combination of ATP-agarose column chromatography and electro-elution was used for its purification from adult female extract. On closer examination, it migrated as a single band at 68 kDa on 10% SDS-PAGE. Peptide sequences TTPSYVAFTDTER, DSGAIAGLNVLR, IINEPTAAAIAYGLDK, NALESYAFNMK and LLSDFFSGK were obtained through MALDI-LC/MS analysis. Confirmation of peptides was accomplished by MASCOT database which showed substantial sequence homology with S. digitata, Wuchereria bancrofti, and Caenorhabditis elegans. Multiple sequence alignment using Clustal W showed 98% identity with W. bancrofti and only 28% with human HSP70. Furthermore, the antigenicity plot has shown that the highly antigenic amino acid residues are constituted within the conserved peptides. These observations suggest a plausible biological connection of ScHSP70 with the disease and its strong immunogenic nature. ScHSP70 showed antigenic cross-reactivity with IgG class of antibody in different categories of filarial sera. However, when IgG subclasses were tested, IgG4 showed high specificity and sensitivity with asymptomatic microfilaraemic sera.

Present report enumerates the vaccine potential of a glutathione-S-transferase purified from Setaria cervi against lymphatic filariasis. In jirds (Meriones unguiculatus) vaccination trial, a very significant 82.75% (p < 0.005) reduction in adult parasite burden was observed in ScGST immunized group after 90 days post Brugia malayi L3 challenge. An inverse correlation between the antibody level and worm burden was found in ScGST immunized group (Person’s correlation r = 0.943, p < 0.05). No recoveries of worms were obtained in heart and lungs of vaccinated group. The Antibodies reactive to ScGST appeared within four weeks of first dose and were able to neutralize the GST activity up to 86%. In an earlier study we have shown vaccine potential of ScGST against B. malayi by ADCC. Evaluation of cytokine profile in T-cells isolated from BALB/c mice immunized with ScGST were also showed predominance of Th2 response which further maintained the humoral immunity generated by ScGST administration in mice. The overall observations prompted us to envisage ScGST as a potential vaccine candidate against lymphatic filariasis.

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.

Background Like other helminth proteases, filarial proteases have also been shown to require for parasite survival inside the host and mediate various physiologic processes such as tissue invasion, feeding, embryogenesis and host immune evasion. Many of these proteases have shown potential for vaccines and chemotherapeutic agents against active filarial infections. Setaria cervi is a bovine filarial parasite and serves as a good parasite model for the studies in lymphatic filariasis. Recently, a 175 kDa collagenase and leucine aminopeptidase (LAP) have been purified and characterized from the bovine filarial parasite S. cervi and shown to be potential vaccine candidate and diagnostic marker, respectively for human lymphatic filariasis. However, their tissue localizations and putative roles in the parasite biology have not yet been examined and thus remain unclear. Therefore, the current study attempts to localize and explore the putative roles of these two enzymes in S. cervi. Methods The tissue distributions of 175 kDa collagenase and leucine aminopeptidase in S. cervi were examined by immunohistochemical and histochemical methods, respectively. Immune sera obtained from the jirds immunized with collagenase served as primary antibody, rabbit anti-mouse IgG-HRP conjugate as secondary antibody and DAB as the substrate for the immunostaining of collagenase. Leu-βNA was used as the substrate for the histochemical staining of LAP. Results Both the collagenase and LAP were present in the body wall; however, they differ in their distribution pattern in different layers of body wall. Collagenase was mainly localized in epicuticle, cuticle, syncytial hypodermis and the nerve cord region whereas LAP was more concentrated in epicuticle, longitudinal muscle layers and almost absent or very faintly stained in syncytial hypodermis and nerve cord region. Both collagenase and LAP showed their common distributions in intestine, uterus and mature eggs, growing embryos and mf. Very strong immunostaining of collagenase in the outer body surface of the parasite indicates its major role in host-parasite relationship whereas the presence of LAP in muscular region suggests its role in tissue remodeling. The common presences of collagenase and LAP in the S. cervi intestine, ovary, uterus, eggs and mf suggest that they also have collaborative roles in molting, nutrition and embryogenesis. The data obtained on their immunological characterizations and their presence in important parasite organs give strong indication that they are critical for the survival of filarial parasite and thus can be good vaccine candidates and/or diagnostic markers for human lymphatic filariasis. Conclusion The manuscript reports for the first time the tissue distribution of collagenase and LAP in the bovine filarial parasite S. cervi and discuss their putative roles in vivo. Our findings also open the avenue to examine the roles of these two proteases in vivo, which will require further experiments like using their natural substrates and/or specific inhibitors in each tissues.

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass approximately = 20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/microg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.

Glutathione-S-transferase (GST) a Phase-II drug detoxification enzyme, was detected in Setaria cervi, a bovine filarial parasite. In vitro effect of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone on the GST of adult female S. cervi was assayed by the addition of these compounds in the maintenance medium. The specific activity of GST towards 1-chloro-2,4-dinitrobenzene was increased progressively 1.2-1.97, 1.3-2.4 and 1.2-2.7 times at 10-100 microM of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone, respectively, after 5 h at 37 degrees C. Substrate specificity studies showed a higher increase in specific activity with ethacrynic acid and no change with cumene hydroperoxide. Although the intensity of GST activity band was more in extract from diethylcarbamazine or butylated hydroxyanisole treated worms extract, an extra band of activity appeared in those worm extracts compared to control worm extract. SDS/PAGE showed increased thickness of the band corresponding to purified GST in extracts from diethylcarbamazine/butylated hydroxyanisole/phenobarbitone treated worms. Purification and quantification of GST from diethylcarbamazine and butylated hydroxyanisole treated worms indicated an increase in enzyme specific activity. The increase in GST protein by these agents was blocked by prior treatment with actinomycin D, indicative of a transcription dependent response. The role of this enzyme in motility and viability of microfilariae and adult female was tested in vitro using a range of known GST inhibitors. Of those tested, ethacrynic acid, ellagic acid, 1-chloro-2,4-dinitrobenzene, cibacron blue and butylated hydroxyanisole reduced the viability and motility of microfilariae and adult female worms at micromolar concentrations. These results suggest that S. cervi GST is inducible in response to the antifilarial drug diethylcarbamazine and may play an important role in parasite's survival, thus could be a potential drug target.

The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.