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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.

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.

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.