Bovine Filarial Parasite Research Articles

Crude protein fraction with high thioredoxin reductase (TrxR) enzyme activity from filarial parasite Setaria cervi counters lipopolysaccharide (LPS)-induced inflammation in macrophages.

Host-parasite interaction has always been an area of interest to the parasite biologists. The complex immune interactions between the parasite and/or the parasite-derived products with the host immune cells determine the fate of the disease biology. Parasitic organisms are widely equipped with a vast array of protective machineries including antioxidant enzymes to withstand the hostile condition inside the host body. The reactive oxygen species (ROS) generated inside the host as a result of parasitic intervention can be endured by the parasite by their own tools to ensure their survival. One such antioxidant enzyme in the filarial parasite that plays a significant role in redox homeostasis, survivability and disease progression is the thioredoxin reductase (TrxR). Herein, we have projected a crude lysate of the bovine filarial parasite Setaria cervi enriched with high TrxR enzyme activity has the capacity to downregulate lipopolysaccharide (LPS)-induced inflammatory macrophages. TrxR-mediated inhibition of the TLR4-NF-κB axis resulting into downregulation of the pro-inflammatory cytokines with concomitant upregulation of the anti-inflammatory cytokines supports the filarial parasite to produce an anti-inflammatory milieu which ultimately promotes worm survivability inside the host and pathogenesis.

Identification of glucose regulated protein94 (GRP94) in filarial parasite S. cervi and its expression under ER stress.

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 91kDa protein was over expressed, after the parasites were maintained in glucose deprived medium. The MALDI- LC/MS analysis of the 91kDa 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 3h and 6h 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 6h 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.

Disruption of redox homeostasis with synchronized activation of apoptosis highlights the antifilarial efficacy of novel piperine derivatives: An in vitro mechanistic approach

A series of novel piperine derivatives were synthesized with high yield and were evaluated for its antifilarial potential against the bovine filarial parasite Setaria cervi. Among 21 (3a-3u) compounds screened, three of them (3k, 3l, 3s) showed significant potential against all the developmental stages (oocytes, microfilariae and adult) of the filarial worm in time and dose dependent manner. 3l showed the highest efficacy among the selected three compounds. These three compounds were further evaluated for both in vitro and in vivo toxicity analyses which further fortified the benign nature of the selected compounds. The antifilarial activities they exhibited were clearly fuelled through disparity of the internal redox homeostasis as evidenced from the alterations in the enzymatic and non-enzymatic antioxidants level which ultimately shifted towards activation of pro-apoptotic signaling cascade eventually leading to the death of the parasites. The ability of the compound 3l to bind thioredoxin reductase and CED-3 protein are the key findings of this study. The present study supported with several biological experiments is therefore a maiden report on the antifilarial effectiveness of these novel piperine derivatives.

Characterization of filarial phosphoglycerate kinase

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-vitro and in silico efficacy of isolated alkaloid compounds from Rauvolfia tetraphylla L. against bovine filarial parasite Setaria cervi: a drug discovery approach.

Bioassay guided isolation from the leaves of Rauvolfia tetraphylla L. resulted in the isolation and characterization of three compounds of alkaloid in nature namely, Curan-17-oic acid (F1); 18, 19-Secoyohimban (F2) and Reserpiline (F3). Macrofilaricidal activity of three compounds was tested against bovine filarial parasite Setaria cervi using in vitro assays and supported by in silico docking analysis on glutathione-S-transferase (GST) enzyme of Wuchereria bancrofti. All the molecules inhibited GST enzyme to some extent 35.78%, 78.22% and 64.21% respectively. Results were supported by molecular docking studies, which showed docking scores for compound F1 (- 5.14), compound F2 (- 7.19) and compound F3 (- 7.2) on GST enzyme. Thus, in conclusion the in vitro and in silico studies indicated that isolated compounds are promising, inexpensive and widely available natural leads, which can be designed and developed into the macrofilaricidal drugs.

Exploring the homolog of a novel proinflammatory microfilarial sheath protein (MfP) of Wuchereria bancrofti in the adult-stage bovine filarial parasite Setaria cervi

A novel microfilarial sheath protein (MfP) of the human filarial parasite Wuchereria bancrofti and its proinflammatory activity on host macrophages were identified recently. MfP is a homolog of the nematode bestrophin-9 superfamily that acts as a ligand of macrophage Toll-like receptor 4 (TLR4) to induce inflammation through NF-κB activation. Therefore, the presence and functional implication of this novel protein in adult-stage parasites were open questions to answer. In this study, the bovine filarial parasite Setaria cervi was used to simulate adult W. bancrofti. We detected the presence of MfP in adult-stage S. cervi through clear immunological cross-reactivity and immunolocalization employing an anti-MfP antibody developed in mice. Therefore, our findings put forward S. cervi as a cost-effective source of immunodominant filarial antigen MfP to simulate its future utilization in the immunotherapeutic intervention of lymphatic filariasis.

Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi.

Antifilarial potential of three medicinal plants namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa was explored using Setaria cervi, a bovine filarial parasite at concentrations of 2.5, 5 and 10mg/ml. Amongst all the extracts, methanol extract of T. bellerica showed highest macrofilaricidal activity i.e. 84.63±1.11 at 10mg/ml in MTT reduction assay with IC50 value of 2.7mg/ml. which was better than the standard DEC i.e. 79.22±3.1% at 10mg/ml with IC50 value 2.84mg/ml. Other plant extracts showed mild in vitro macrofilaricidal activity. T. bellerica methanol extract exhibited significant GST activity of 18.86±0.21 and 12.83±0.03μM/ml/min at 5 and 10mg/ml with percentage inhibition value of 73.96% and 82.29% respectively. DEC showed GST activity value of 40.03±4.14 and 21.48±6.44μM/ml/min with percentage inhibition value of 21.76% and 58.01% at 5 and 10mg/ml respectively. Thus, methanol extract of leaves of T. bellerica exhibited highly significant antifilarial potential and needs detailed analysis.

Identification and characterization of a novel prolyl oligopeptidase in filarial parasite Setaria cervi

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.

Thioredoxin reductase from the bovine filarial parasite Setaria cervi: Studies on its localization and optimization of the extraction

Exploration of novel drug targets has been the major thrust area in filarial research. In this regard, identification and characterization of oxidative enzymes that play pivotal role in the survival of filarial parasite inside host are of immense importance. In this study, we are reporting the presence of an important redox regulatory enzyme, thioredoxin reductase (TrxR) in the bovine filarial parasite Setaria cervi. TrxR was found to be exists throughout the developmental stages viz. oocyte, microfilaria and adult of the parasite. Since further studies on this enzyme require adequate quantity, influential extraction parameters were optimized statistically using response surface methodology (RSM) employing a seven factors based Box-Behnken design matrix. ANOVA analysis revealed the relative importance of each parameter and a regression equation was eventually developed that could predict the specific activity (SA) of TrxR. Finally the optimized extraction conditions predicted by RSM was 6.1ml of 61.86mM buffer, pH 6.0, with extraction temperature 39.96°C for 180min in addition to 450rpm agitation and 20μl/ml of protease inhibitor. Therefore this study is going to be the maiden report depicting the identity of TrxR in filarial parasite and the optimized extraction conditions for its isolation with better kinetic efficiency.

Characterization of immunoreactive proteins of Setaria cervi isolated by preparative polyacrylamide gel electrophoresis.

Filarial parasites are complex mixtures of antigenic proteins and characterization of these antigenic molecules is essential to identify the diagnostically important filaria-specific antigens. In the present study, we have fractionated the somatic extracts from adults of Setaria cervi (bovine filarial parasite) on preparative SDS-polyacrylamide gel electrophoresis and tested the immunoreactivity of the separated gel fractions with polyclonal antibodies against filarial excretory-secretory antigens as well as filarial patients sera. The SDS-PAGE analysis of gel eluted fractions revealed 1 protein band in F-1 fraction, 2 protein bands in F-2 fraction and 2-3 protein bands in all other fractions (F3- F11). Seven gel eluted fractions (F1, F2, F3, F4, F5, F6 and F11) showed high ELISA reactivity with the polyclonal antibody (against excretory-secretory antigen) and four of these fractions (F-1, F-2, F3 and F6) exhibited high ELISA reactivity with antibodies present in filarial patient sera. The reactivities of the gel fractions (F1 and F2), recognized by filarial patients sera, were also tested with the monoclonal antibody (detecting the filarial circulating antigen). The F1 and F2 gel eluted fractions were found to have the target antigen of monoclonal antibody as evident by high reactivity with the monoclonal antibody in ELISA and immunoblotting. The S. cervi gel eluted F1 fraction (containing single antigen) could detect antibodies in filarial patients sera and not in non-filarial sera thereby suggesting its usefulness for specific serodiagnosis of human filariasis.

C-cinnamoyl glycosides as a new class of anti-filarial agents

A series of C-cinnamoyl glycosides has been synthesized in good yield by the BF3·OEt2 catalyzed aldol condensation of C-glycosylated acetone derivative with a variety of aromatic aldehydes. The synthesized compounds were evaluated for their potential as anti-filarial agents against bovine filarial parasite Setaria cervi and human filariid Wuchereria bancrofti using a number of biological assays such as relative movability (RM) assessment and MTT reduction assay. Among twenty seven test compounds six compounds were found active in terms of MIC, IC50 and LC50 values. Further biological studies were carried out using three lead compounds because of their significantly low MIC values and IC50 values compared to the standard anti-filarial drug Ivermectin. In addition, structure activity relationship study of the test compounds has been carried out using 3D-QSAR analysis.

In search of a potential diagnostic tool for molecular characterization of lymphatic filariasis.

Lymphatic filariasis (LF) is a chronic disease and is caused by the parasites Wuchereria bancrofti (W. bancrofti), Brugia malayi (B. malayi) and Brugia timori (B. timori). In the present study, Setaria cervi (S. cervi), a bovine filarial parasite has been used. Previously, it has been reported that the S. cervi shares some common proteins and antigenic determinants with that of human filarial parasite. The larval stages of filarial species usually cannot be identified by classical morphology. Hence, molecular characterization allows the identification of the parasites throughout all their developmental stages. The genomic DNA of S. cervi adult were isolated and estimated spectrophotometrically for the quantitative presence of DNA content. Screening of DNA sequences from filarial DNA GenBank and Expressed Sequence Tags (EST's) were performed for homologous sequences and then multiple sequence alignment was executed. The conserved sequences from multiple sequence alignment were used for In Silico primer designing. The successfully designed primers were used further in PCR amplifications. Therefore, in search of a promising diagnostic tool few genes were identified to be conserved in the human and bovine filariasis and these novel primers deigned may help to develop a promising diagnostic tool for identification of lymphatic filariasis.

Evidence of reactive oxygen species (ROS) mediated apoptosis in Setaria cervi induced by green silver nanoparticles from Acacia auriculiformis at a very low dose

Green synthesis of silver nanomaterial plays a pivotal role in the growing field of nanotechnology. Development of anti-parasitic drugs from plant metabolites has been in regular practice from the ancient period but most of them were discarded due to their inefficiency to control diseases effectively. At present, nanoparticles are used for developing anti-parasitic therapy for their unique properties such as smallest in size, bio-ability, bio-compatibility and penetration capacity into a cell. The present study aims at synthesis of silver nanoparticles (AgNPs) by using funicles extract of Acacia auriculiformis and tests its efficacy as antifilarial. Experimental evidence show that AgNPs are effective at a very low concentration compared to crude plant extracts. Synthesis of these nanoparticles is a single-step, biogenic, cost effective and eco-friendly process. Synthesized nanoparticles were characterized by UV–Vis spectroscopy, TEM, SAED, FTIR, EDX, FESEM and Z-potential. The antifilarial efficacy of AgNPs was tested against different life cycle stages of bovine filarial parasite Setaria cervi by morphological study, motility assessment and viability assay. These nanoparticles are found to have antifilarial activity with LC50 of 5.61 μg/mL and LC90 of 15.54 μg/mL against microfilaria of S. cervi. The microscopic findings and the detailed molecular studies confirmed that green synthesized AgNPs were effective enough to induce apoptosis through up regulation of ROS (reactive oxygen species).

Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

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 21kDa 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.

Isolation and characterization of endochitinase and exochitinase of Setaria cervi

Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65–70kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18μM.

Identification of a novel stress regulated FERM domain containing cytosolic protein having PTP activity in Setaria cervi, a bovine filarial parasite

A 67 kDa cytosolic FERM domain containing protein having significant protein tyrosine phosphatases activity (PTPL) has been purified to homogeneity from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 16 peptide peaks showing nearest match to Brugia malayi Moesin/ezrin/radixin homolog 1 protein and one peptide showing significant similarity with a region lying in the catalytic domain of human PTPD1. PTPL showed significant cross reactivity with the human PTP1B antibody and colocalize with actin in the coelomyrian cells of hypodermis in the parasite. PTPL was stress regulated as it showed marked decrease in the expression when exposed to Aspirin, an antifilarial drug and Phenylarsine Oxide, PTP inhibitor.

Effect of CDNB on filarial thioredoxin reductase : A proteomic and biochemical approach.

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.

Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

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.

Modulation of phosphofructokinase (PFK) from Setaria cervi, a bovine filarial parasite, by different effectors and its interaction with some antifilarials

BackgroundPhosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux. This enzyme has been extensively studied from mammalian sources but relatively less attention has been paid towards its characterization from filarial parasites. Furthermore, the information about the response of filarial PFK towards the anthelmintics/antifilarial compounds is lacking. In view of these facts, PFK from Setaria cervi, a bovine filarial parasite having similarity with that of human filarial worms, was isolated, purified and characterized.ResultsThe S. cervi PFK was cytosolic in nature. The adult parasites (both female and male) contained more enzyme activity than the microfilarial (Mf) stage of S. cervi, which exhibited only 20% of total activity. The S. cervi PFK could be modulated by different nucleotides and the response of enzyme to these nucleotides was dependent on the concentrations of substrates (F-6-P and ATP). The enzyme possessed wide specificity towards utilization of the nucleotides as phosphate group donors. S. cervi PFK showed the presence of thiol group(s) at the active site of the enzyme, which could be protected from inhibitory action of para-chloromercuribenzoate (p-CMB) up to about 76% by pretreatment with cysteine or β-ME. The sensitivity of PFK from S. cervi towards antifilarials/anthelmintics was comparatively higher than that of mammalian PFK. With suramin, the Ki value for rat liver PFK was 40 times higher than PFK from S. cervi.ConclusionsThe results indicate that the activity of filarial PFK may be modified by different effectors (such as nucleotides, thiol group reactants and anthelmintics) in filarial worms depending on the presence of varying concentrations of substrates (F-6-P and ATP) in the cellular milieu. It may possess thiol group at its active site responsible for catalysis. Relatively, 40 times higher sensitivity of filarial PFK towards suramin as compared to the analogous enzyme from the mammalian system indicates that this enzyme could be exploited as a potential chemotherapeutic target against filariasis.

Surface of the filarial parasite Setaria digitata and the structural changes on treatment with Triton X-100

Abstract Setaria digitata, a bovine filarial parasite is used as a laboratory model for filariasis. The studies showed that considerable amount of biochemically and immunologically active materials are present on the surface of the parasite. The effect of Triton X-100 on the surface of the parasite, when it is used to separate the surface associated materials, is not known. Treatment with Triton X-100 enhances the amount of ES protein released into the medium. The electron microscopic study of S. digitata wall showed that structure of the surface is similar to that of W. bancrofti. There are striations, annulations and button like bosses on the filarial surface. On treatment with Triton X-100 the electron dense cloudy layer on the surface was disappeared. The surface appeared to be suitable for hosting secreted molecules on the surface.