Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

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.     

Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females 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 against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, 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.     

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.     

Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi

The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources.     

Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi

The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.     

Brugia malayi: recombinant antigens expressed by genomic DNA clones

A Brugia malayi genomic DNA expression library was screened with rabbit antiserum generated against live infective larvae and 33 clones were identified. Five randomly selected clones were characterized in detail by Western blot, DNA and RNA analyses. The fusion proteins produced by each of these recombinant DNA clones are expressed by different genomic sequences. A profile of antigenic cross-reactivities of all 33 recombinant clones was compiled using a battery of antisera, including sera from humans infected with B. malayi. A high percentage of clones were cross-reactive with antisera against the filarial parasites B. pahangi, Dirofilaria immitis, and Onchocerca volvulus. We have made a preliminary identification of three categories of recombinant clones encoding (1) antigens that were cross-reactive with some or all antisera tested, (2) antigens that were specific to the Brugia genus, and (3) antigens that appeared to be specific to B. malayi. These recombinant antigens are candidates for further studies in filarial immunoprophylaxis and diagnosis.     

Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. Setaria cervi, the causal organism of setariasis and lumbar paralysis in cattle, is routinely employed as a model organism for conducting biochemical and enzymatic studies on filarial parasites. In view of the practical difficulties in procuring human strains of Wuchereria bancrofti and Brugia malayi for drug screening, the bovine filarial parasite S. cervi, resembling the human species in having microfilarial periodicity and chemotherapeutic response to known antifilarial agents, is widely used as a model in such studies. For a rational approach to antifilarial chemotherapy, knowledge of the biochemical composition and metabolic pathways of this helminth parasite may be of paramount importance, so that more potent antifilarial agents based on specific drug targets can be identified in drug discovery programmes. The present review provides an update on the biochemistry of the important metabolic pathways functioning within this potentially important bovine parasite, that have so far been studied, and on those that need to be investigated further so as to identify novel drug targets that can be exploited for designing new antifilarial drugs.     

Characterization of Ca(2+)-ATPase of Setaria cervi (Nematoda: Filarioidea): effect of phenothiazines and anthelmintics.

1. A Mg2+ independent, Ca(2+)-ATPase requiring high concentrations of Ca2+ (5 mM) for the activation, equally distributed in cuticle-muscular-hypodermis, genital organs and gastrointestinal tissues and mainly localized in 10,000 g pellet fraction, was identified in Setaria cervi, a bovine filarial parasite. 2. Filarial enzyme showed Km value of 3.33 mM for ATP as computed from the double reciprocal Lineweaver-Burk plot. 3. The enzyme could be completely solubilized by sonication with about 4-fold increase in specific activity of the enzyme. 4. The enzyme showed about 2-fold activation by the calmodulin fractions isolated from S. cervi and rat brain homogenates. 5. The enzyme was highly sensitive to inhibition with some phenothiazine derivatives. Trifluoperazine was observed to be the most potent inhibitor followed by promethazine and chlorpromazine. 6. Some anthelmintics viz. diethycarbamazine and centperazine were found to be highly potent inhibitors of the enzyme, significant inhibition of filarial Ca(2+)-ATPase was also observed with levamisole and suramine. 7. Studies indicate Ca(2+)-ATPase of S. cervi as a potential chemotherapeutic target.     

Expansion of Foxp3+ regulatory T cells in mice infected with the filarial parasite Brugia malayi

Many helminths, including Brugia malayi, are able to establish long-lived infections in immunocompetent hosts. Growing evidence suggests that the immune system's failure to eliminate parasites is at least partially due to the effects of regulatory T cells (Tregs). To test whether parasites may directly stimulate host regulatory activity, we infected mice with two key stages of B. malayi. Both mosquito-borne infective larvae and mature adults i.p. introduced were found to preferentially expand the proportion of CD25(+)Foxp3(+) cells within the CD4(+) T cell population. The induction of Foxp3 was accompanied by raised CD25, CD103, and CTLA-4 expression, and was shown to be an active process, which accompanied the introduction of live, but not dead parasites. CTLA-4 expression was also markedly higher on Foxp3(-) cells, suggesting anergized effector populations. Peritoneal lavage CD4(+)CD25(+) cells from infected mice showed similar suppressive activity in vitro to normal splenic "natural" Tregs. Both B. malayi larvae and adults were also able to induce Foxp3 expression in adoptively transferred DO11.10 T cells, demonstrating that filarial infection can influence the development of T cells specific to a third party Ag. In addition, we showed that induction was intact in IL-4R-deficient animals, in the absence of a Th2 or alternatively activated macrophage response. We conclude that filarial infections significantly skew the balance of the host immune system toward Treg expansion and activation, in a manner dependent on live parasites but independent of a concomitant Th2 response.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

A major allergen of lymphatic filarial nematodes is a parasite homolog of the gamma-glutamyl transpeptidase.

BACKGROUND: Bm2325, a major IgE-inducing antigen of the filarial parasite Brugia malayi has been implicated in the pathology of tropical pulmonary eosinophilia (TPE), a pulmonary syndrome thought to result from hypersensitivity to microfilariae. MATERIALS AND METHODS: Affinity-purified IgE to Bm2325 from patients with TPE was used to identify a complementary DNA (cDNA) from a B. malayi expression library. Sequence analysis of the cDNA revealed a hitherto unknown parasite protein. Immunoblotting of the recombinant filarial protein using sera of patients with TPE determined its IgE-binding capacity. Reactivity to human lung epithelial cell proteins was analyzed using murine anti-Bm2325 antibodies and serum from patients with TPE. RESULTS: The predicted protein is a homolog of the entire precursor of the gamma-glutamyl transpeptidase (gamma-GT), a key enzyme in the synthesis and degradation of glutathione. The filarial precursor encodes both the heavy (H) and the light (L) chain subunits and shares structural similarities with the mammalian enzymes. The Bm2325 allergen was identified as the homolog of the enzyme light chain subunit. Murine antibodies against the recombinant parasite gamma-GT cross-reacted with the human enzyme present in human airway epithelial cells, and human gamma-GT is a target of antibodies present in the serum of patients with TPE. CONCLUSION: Molecular mimicry between the parasite gamma-GT homolog and the host membrane-bound gamma-GT present in lung epithelial cells likely contributes to the pathogenesis observed in tropical pulmonary eosinophilia.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Significance of transglutaminase-catalyzed reactions in growth and development of filarial parasite, Brugia malayi

A novel form of transglutaminase enzyme [EC 2.3.2. 13] was identified in adult worms of Brugia malayi. The molecular size of this enzyme was 22-kilodaltons as determined by Western blot and immunoprecipitation, using a monoclonal (CUB 7401) or polyclonal antibodies against guinea-pig liver tissue transglutaminase. The enzyme was present in female worms only; adult males contained no detectable levels of the enzyme peptide. Possible involvement of transglutaminase-catalyzed reactions in growth and survival of filarial parasites was studied by using various enzyme-specific pseudosubstrates. Presence of these inhibitors resulted into a significant inhibition of microfilariae production and release by gravid female worms in a dose-dependent manner. These results suggest that transglutaminase-catalyzed reactions are essential for development of in utero growing embryos to mature microfilariae.     

Identification and cloning of a novel tetraspanin (TSP) homologue from Brugia malayi.

This is the first report of a tetraspanin (TSP)-like molecule in the lymphatic filarial parasites. Expressed sequence tag (EST) database search for TSP like molecules in the filarial genome resulted in three significant EST hits (two partial ESTs from Brugia malayi and one full length EST from Wuchereria bancrofti). The full length gene cloned from B. malayi showed significant similarity to Caenorhabditis elegans TSP and human TSP and hence the gene was named B. malayi TSP (BmTSP). Subsequent Genbank analysis with the predicted ORF of BmTSP showed additional homologous genes reported from Schistosoma mansoni and Taenia solium parasites. Structural analyses showed that BmTSP has four transmembrane domains and other conserved domains such as CCG and two other critical cysteine residues present within the large extracellular loop similar to other reported TSPs. In addition, putative post-translational modifications such as N-glycosylation, protein kinase c phosphorylation, casein kinase II phosphorylation and N-myristoylation sites have been found in BmTSP sequence. Further, PCR analyses showed that BmTSP is differentially transcribed, with highest level of expression being present in the adult stages followed by L3 and mf stages. This study thus describes a novel TSP cloned from B. malayi, its putative functions in cuticle biogenesis and role in protective immunity.     

In vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines: a new class of antifilarial agents

A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of these, six compounds (4a-f) possessed improved in vitro anti-filarial activity and examples 4d and 4f were also found to be active in the in vivo experiments. These results demonstrate that 3,6-epoxy [1,5]dioxocines exhibits potent antifilarial activity and might be developed into a new class of antifilarial drug.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

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.     

Brugia malayi: transient transfection by microinjection and particle bombardment

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.     

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.