An ARV1 homologue from a filarial nematode is functional in yeast.

The transmembrane protein, ARV1, plays a key role in intracellular sterol homeostasis by controlling sterol distribution and cellular uptake. To date, only the ARV1s from yeast and humans have been characterized to some extent. In this study, the ARV1 of an animal filarial parasite, Setaria digitata (SdARV1), was characterized; its cDNA was 761 bp and encoded a protein of 217 amino acids, with a predicted molecular weight of 25 kDa, containing a highly conserved ARV1 homology domain and three transmembrane domains in the bioinformatic analyses. Information required to cluster members belonging to a particular taxon has been revealed in phylogenetic analyses of ARV1 sequences derived from different organisms. Reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that SdARV1 was expressed in different developmental stages - microfilariae and adult male and female worms. Experiments carried out with a single copy of the SdARV1 under the control of the PMA-1 promoter in a temperature-sensitive Saccharomyces cerevisiae mutant strain indicated full complementation of the mutant phenotype, with growth at a non-permissive temperature (37°C). Microscopic observations of cellular morphology with Gram staining revealed alteration of the shape from shrunken to oval, in mutant and complemented strains, respectively. Assessment of free sterol levels extracted from mutant yeast and complemented strains indicated that the level of sterol was significantly higher in the former compared to the latter, which had sterol levels similar to those of the wild type. Thus, the results of the current study suggest that SdARV1 is ubiquitously expressed in different developmental stages of S. digitata, and that it is a true functional homologue of mammalian and yeast ARV1s, which have crucial phylogenetic information that follows classical evolutionary trends. Finally, this is the first study to report the biological function of nematode ARV1.

Development of siRNA mediated RNA interference and functional analysis of novel parasitic nematode-specific protein of Setaria digitata.

Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.

Detection for cross-reactive proteins in filarial worm Setaria equina, MCF-7 human breast cancer, and Huh-7 hepatoma cells.

This study aimed to detect the cross-reactive proteins in filarial parasite adult worm Setaria equina and two different tumor cell lines (MCF-7 human breast cancer and Huh-7 hepatoma cells). This was performed using rabbit anti-S. equina extract (SeqE) or DEC (Diethylcarbamazine citrate) polyclonal IgG antibodies by indirect ELISA and western blotting. The results indicated cross-reactive bands at 70 and 75 kDa in all extracts by anti-DEC and SeqE antibodies, respectively. In addition, the expression of 70 kDa protein was only reduced in filarial worms and Huh-7 after in vitro DEC treatment compared to the control.

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 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.

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 diethylcarbamazine citrate and Setaria equina excretory-secretory material on rat hepatocellular carcinoma.

Diethylcarbamazine citrate (DEC) has been known for its efficacy to eradicate bancroftian filariasis in Egypt and other countries in the world. One of the known effects was to decrease the level of circulating filarial antigen in the patient's serum. The target of this study was to examine the effect of DEC, excretory-secretory (ES) material from the filarial parasite Setaria equina or a combination of both on the status of oxidative stress and pathogenesis of rat hepatocellular carcinoma (HCC) induced by diethylnitrosamine and 2-acetylaminofluorene. This could be tested in vitro using nitroblue tetrazolium reduction test for measuring the level of superoxide anion (O2(•-)) released from rat peritoneal macrophages. For in vivo test, a single dose before induction of carcinogenesis or continually repeated doses with DEC, ES or DEC + ES was used. Exposure of macrophages to ES could lead to a significant decrease (p < 0.01) in O2(•-) release, while DEC (200 µM) could modulate such effect with significant increase (p < 0.05). Pathogenesis of liver cancer and treatment were evaluated using histological investigation, level of antioxidant and liver function enzymes. Repeated ES doses could increase the activity of antioxidant enzymes, especially the catalase enzyme and show a protective effect on liver architecture. DEC could modulate the later effects when combined with ES. No significant effect on the liver function enzymes after treatment was observed. Nuclear factor κB was found to be localized only in the cytoplasm after single and repeated treatments with ES. This study could indicate the effect of S. equina ES as antioxidant against rat HCC, while DEC could modulate such effect when combined with it.

Novel parasitic nematode-specific protein of Setaria digitata largely localized in longitudinal muscles, reproductive systems and developing embryos.

Parasitic nematodes may have common properties in parasitizing the host which are conferred by related parasitic proteins encoded by their genome. A novel protein characterized from bovine filarial nematode Setaria digitata was found to be present only in the parasitic nematodes and expressed at all the stages of the nematode's life. In immunohistochemical staining using polyclonal antibodies prepared against recombinant S. digitata protein, the highest expression of S. digitata novel protein (SDNP) was seen in the longitudinal muscles of the body wall of adult males and females indicating its possible involvement in parasite locomotion. Moderate expression was observed in the reproductive organs of both sexes while showing gradual increase in the expression as the development of the reproductive tissue progressed suggesting its role in tissue transformation in male and female reproduction. A low level of expression was observed in the cuticle, syncytial hypodermis region, lateral line and the intestinal wall. Further, the expression of SDNP was also seen in developing microfilaria within the uterus of female worms, developing spermatozoa of males and different developmental stages of embryos implicating its involvement in nematode growth and development. Subcellular localization of SDNP carried out in yeast, Pichia pastoris using green fluorescence construct revealed that this protein localized mainly in the nucleus and partly in the cytoplasm. Comprehensive bioinformatics analyses indicated that this protein contains a nuclear localization signal, RNAP_Rpb7_N_like domain, regions that are homologous to a part of the nuclear factor localization-like domain, interdomain linkers of muscle specific twitchin kinase of Caenorhabditis elegans and calcium-dependent protein kinase isoform CDPK1 of Arabidopsis thaliana. Therefore, considering all the outcomes together, it can be suggested that the SDNP is a parasitic nematode-specific, nuclear and cytoplasmic protein that is likely to be regulated by reversible phosphorylation-dephosphorylation reaction, expressed in all the stages of nematode's life having pivotal functional roles in muscle, reproductive systems, embryogenesis, and also in the growth and development.

Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata.

OBJECTIVE: To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses. METHODS: To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently. RESULTS: Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa. CONCLUSIONS: SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

Setaria digitata: identification and characterization of a hypodermally expressed SXP/RAL2 protein.

SXP-1, an immunodominant filarial protein identified from Wuchereria bancrofti from our centre and previously exploited for diagnosis of human lymphatic filariasis, has been shown to be well conserved across several filarial species. In the present study, we describe the identification of SXP protein from the cattle filarid Setaria digitata using antiserum raised against recombinant WbSXP-1, and were able to detect 34 and 66kDa proteins from the crude protein extracts of S. digitata. These reactive proteins were found to be sheath proteins localized to the hypodermal region of the parasite.

Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes.

Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(beta1-3)Man(beta1-4)Glc(1-1)ceramide, GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta1-4)Glc(1-1) ceramide and Gal(alpha1-3)GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta 1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C(22h:0)), tricosanoic (C(23h:0)) and tetracosanoic (C(24h:0)) acids, and C(17) sphingosine (C(d17:1)) (where (h) is hydroxylated and (d) is dihydroxylated).

Molecular characterisation of a hsp70 gene from the filarial parasite Setaria digitata.

The filarial parasite Setaria digitata is the causative agent of cerebrospinal nematodiasis in its abnormal hosts such as sheep, goats and horses, and therefore is of significant veterinary importance. Since very little is currently known about the biology of this parasite at molecular level, we have cloned and characterised a hsp70 gene, the first gene to be reported from this parasite. The genomic clone isolated contained sequences from two hsp70 genes. One gene, hsp70-2, was completely sequenced and found to contain nine introns ranging in size from 78 to 195 bp. The region upstream of the initiation codon contained a putative TATA box, two CAAT box elements and three heat-shock elements. A putative transcription initiation site was also identified. The 5' untranslated region contained a splice acceptor sequence. The gene was typically AT rich, having a GC content of 44.5%. The deduced aa sequence potentially encoded a cytosolic protein of 645 aa, which had three consecutive repeats of a tetrapeptide motif, GGMP, at the carboxyl end. The gene appeared to be constitutively transcribed and was not significantly enhanced in response to heat shock in adult worms. Another hsp70 gene (hsp70-1) was located further upstream, arranged in direct tandem with hsp70-2. Southern blot analysis revealed the presence of two or three additional hsp70-related genes in the S. digitata genome.

A sensitive polymerase chain reaction based assay for the detection of Setaria digitata: the causative organism of cerebrospinal nematodiasis in goats, sheep and horses.

A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previously reported oligonucleotide based chemiluminescent detection of microfilariae in infected host blood samples and L3 larvae in mosquitoes.

Apoptosis induced by filarial parasitic sheath protein in HEp 2 cell lines blocked by ectopic expression of bcl 2.

Chronic filarial patients exhibit an occult manifestation, Tropical Pulmonary Eosinophilia, (TPE), caused by an exaggerated immune response to shed and circulating filarial antigens, leading to extensive lung damage. We have attempted to examine the disease in vitro using the human epithelial cell line, HEp2. Filarial sheath proteins induce apoptosis in HEp2 cells characterized by chromatin condensation, internucleosomal DNA cleavage, positive staining for TUNEL assay and shows a sub-G1 peak on FACS analysis. In order to understand subcellular events and to analyse the protective role of bcl2, we engineered HEp2 to overexpress Bcl2 protein. HEp2 bcl2 cells do not undergo apoptosis on exposure to filarial sheath protein, indicating that filarial protein-induced apoptosis in epithelial cells proceeds via a pathway, inhibitable by overexpression of bcl 2.

Necrosis of lung epithelial cells by filarial parasitic protein via an early induction of c-H-ras and TNF alpha expression.

The direct interaction of filarial proteins with lung epithelial cells was examined to determine the possible mechanism of inducing cell death, an event that is observed in patients with tropical pulmonary eosinophilia. Exposure of lung epithelial cells to filarial parasitic proteins, Brugia malayi (BmA), Setaria digitata (Sd), and recombinant filarial protein (pGT 7) in vitro for more than 2 days, causes the appearance of DNA fragments both in the cytoplasm and culture supernatants, while no fragmentation was observed in the untreated controls. The release of DNA fragments both in the cytoplasm and the culture supernatants simultaneously, indicates that cell death is induced by a necrotic event rather than apoptosis. Fluorescent-labelled studies also indicate the fragmentation of DNA increasing in a time-dependent manner. Normal cellular function is controlled through several oncogenes. The modulation of specific proto-oncogenes like myc, ras and TNF alpha during exposure to filarial parasitic proteins reveal elevated levels of expression of ras and TNF alpha as early as 2 hours, implicating their involvement prior to DNA fragmentation leading to pathogenesis.

Classification of Setaria worms from calves and adult cattle into Setaria digitata and Setaria marshalli by polyacrylamide gel electrophoresis.

The protein profiles of solubilized whole worms of the species, Setaria digitata and Setaria marshalli were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that one band with a molecular size of 69 kDa was confirmed only in S. marshalli, while this band was not detected in S. digitata. There were no differences of the major bands between males and females of the respective worm species. As further investigation, two-dimensional gel electrophoresis was performed and at least one spot ranging from 64 to 73 kDa was confirmed in S. marshalli but not in S. digitata. Therefore, those worms could be classified into two species biochemically as well as by the morphological characteristics.

[Studies on amino acid and chemical element in five species of filariae].

Five species of human and domestic animal filariae (B. m, S. d, S. l, S. e, D. i) after lyophilization and hydrolysis were resolved using HPLC and type WFX-IB AAS to determine amino acid (AA) and microelement (ME) content, respectively. The results showed that there were 16 and 17 AA in B. malayi and the animal filariae, respectively. Among AA, the total amount (microgram/mg dry wt.) was markedly higher in animal filariae than that in B. malayi in which the content of acidic and aliphatic AA was significantly higher than those of basic and aromatic AA, respectively. Among five ME (Zn, Cu, Fe, Mn, Cd) and two microelements (Ca, Mg) found in all these filariae. Zn Content was the predominant ME and Ca was much more than Mg.