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.

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown.

Human bancroftian filariasis - a role for antibodies to parasite carbohydrates.

Studies on immune responses to parasites have been undertaken in filariasis with a view to understand protective immunity, pathogenesis of the disease process and mechanisms of immune deviation. However none of the investigations conducted so far on antibody responses have addressed the issue of immunogenicity of filarial carbohydrate antigens in human lymphatic filariasis. In this communication we report details on relative protein and carbohydrate contents of various developmental stages of filarial parasites and antibody responses to filarial proteins (Fil.Pro) and carbohydrates (Fil.Cho) in different clinical spectrum of human bancroftian filariasis. As expected, antibodies of IgM and IgG2 subclass recognized primarily Fil.Cho while IgG4 filarial antibodies recognized exclusively Fil.Pro. Reactivity of IgG3 to Fil.Cho was similar to that of IgG2 while IgG1 more readily recognized Fil.Pro than Fil.Cho. The IgG2 and IgG3 antibodies to Fil.Cho were found to be significantly more in patients with chronic filarial disease and in endemic normals when compared with microfilariae (mf) carriers while IgG4 antibodies to Fil.Pro were significantly more in mf carriers. The dichotomy in reactivity of filarial IgG2, IgG3 and IgG4 was dependent on active filarial infection as indicated by presence of circulating filarial antigen (CFA). Individuals with CFA were found to possess significantly more IgG4 to Fil.Pro than those without CFA while IgG2 and IgG3 levels to Fil.Cho was significantly more in CFA negative subjects when compared to those with CFA. Although IgG1 reacted more readily with Fil.Pro, unlike IgG4, their levels were significantly more in CFA negative subjects when compared to those with active filarial infection. Absorption of sera with phosphorylcholine (PC) resulted in no significant loss of reactivity to Fil.Cho indicating that most of the anticarbohydrate antibodies were recognizing non-PC determinants in human filariasis. Elevated levels of IgG2 and IgG3 antibodies to Fil.Cho in individuals free of filarial infection indicate a possible role for carbohydrate antigens in induction of protective immunity in human filariasis.

Identification of tgh-2, a filarial nematode homolog of Caenorhabditis elegans daf-7 and human transforming growth factor beta, expressed in microfilarial and adult stages of Brugia malayi.

A novel member of the transforming growth factor beta (TGF-beta) family has been identified in the filarial nematode parasite Brugia malayi by searching the recently developed Expressed Sequence Tag (EST) database produced by the Filarial Genome Project. Designated tgh-2, this new gene shows most similarity to a key product regulating dauer larva formation in Caenorhabditis elegans (DAF-7) and to the human down-modulatory cytokine TGF-beta. Homology to DAF-7 extends throughout the length of the 349-amino-acid (aa) protein, which is divided into an N-terminal 237 aa, including a putative signal sequence, a 4-aa basic cleavage site, and a 108-aa C-terminal active domain. Similarity to human TGF-beta is restricted to the C-terminal domain, over which there is a 32% identity between TGH-2 and TGF-beta1, including every cysteine residue. Expression of tgh-2 mRNA has been measured over the filarial life cycle. It is maximal in the microfilarial stage, with lower levels of activity around the time of molting within the mammal, but continues to be expressed by mature adult male and female parasites. Expression in both the microfilaria, which is in a state of arrested development, and the adult, which is terminally differentiated, indicates that tgh-2 may play a role other than purely developmental. This is consistent with our observation that TGH-2 is secreted by adult worms in vitro. Recombinant TGH-2 expressed in baculovirus shows a low level of binding to TGF-beta-receptor bearing mink lung epithelial cells (MELCs), which is partially inhibited (16 to 39%) with human TGF-beta, and activates plasminogen activator inhibitor-1 transcription in MELCs, a marker for TGF-beta-mediated transduction. Further tests will be required to establish whether the major role of B. malayi TGH-2 (Bm-TGH-2) is to modulate the host immune response via the TGF-beta pathway.

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.

The peroxidoxin 2 protein of the human parasite Onchocerca volvulus: recombinant expression, immunolocalization, and demonstration of homologous molecules in other species.

The peroxidoxin protein of the filarial parasite Onchocerca volvulus (OvPXN-2) belongs to a group of highly conserved antioxidant molecules. For a more detailed characterization of this protein and for determination of its expression pattern the OvPXN-2 protein was recombinantly expressed as a His-tagged protein. Under reducing conditions the recombinant protein had an apparent molecular mass of 28 kDa. Considering the size of the His-tag and the FLAG epitope introduced to the recombinant protein, this size is in agreement with that of the native protein identified in O. volvulus extract. Antiserum raised against the recombinant protein was used for immunolocalization. In O. volvulus the antigen is predominantly expressed in the hypodermis and particularly the lateral and median chords show high levels of expression. The protein is also expressed strongly in the hypodermis of infective larvae and more weakly in microfilariae. Related cross-reacting proteins were detected in several Onchocerca species and other filariae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with Western blotting revealed proteins with almost identical mobility in extracts prepared from O. ochengi, O. gibsoni, and Dirofilaria immitis.

Brugia malayi: resistance of cuticular lipids to oxidant-induced damage and detection of alpha-tocopherol in the neutral lipid fraction.

We have examined the susceptibility of cuticular membrane lipids of Brugia malayi to oxidants generated in vitro. Live parasites as well as extracted cuticular lipids were treated with hydrogen peroxide and hypochlorous acid and the extent of lipid peroxidation was quantified. The cuticular membranes of B. malayi were found to be resistant to lipid peroxidation at hydrogen peroxide concentrations which were lethal to the organism. This resistance was partly due to the inherently low unsaturation indices of the fatty acyl residues, but complete protection was afforded by lipid-soluble antioxidants present in the neutral lipid fraction of the parasites. We have identified alpha-tocopherol as a major antioxidant present in both adult and microfilarial B. malayi. In addition, we report that although hypochlorous acid chemically modifies isolated parasite lipids, the latter do not appear to be the primary substrate for the oxidant in live worms. The data are discussed in terms of the susceptibility of B. malayi to products of the respiratory burst from activated myeloid cells.

Interaction of filarial proteins on growth regulation of normal lung epithelial cells in vitro.

An in vitro model to examine the effects of filarial proteins on lung epithelial cells has been developed. Several of these proteins appear in circulation of infected individuals. A close association between tropical pulmonary eosinophilia (TPE) and filariasis has been reported by several workers. [3H]-thymidine studies do indicate that when optimum concentration of these filarial proteins were added to lung cultures in proliferating and basal/maintenance media a further increase in growth stimulation was observed early in culture. However, on longer exposures and at higher concentrations an inhibitory effect with distinct morphological changes were noted. The dual role of these proteins on lung epithelial cells in vitro may highlight the possibility of a direct interaction of these proteins with lung cells during disease also contributing to tissue damage.

Cytochemical analysis of the sheath of microfilariae of Wuchereria bancrofti and Brugia malayi.

Thin sections of Epon and Lowicryl embedded microfilariae of Wuchereria bancrofti and Brugia malayi were analyzed by transmission electron microscopy aiming at topochemical characterization of the sheath. Three layers could be distinguished. Some of the layers were labeled when incubated in the presence of antibodies, lectins and enzymes which recognize extracellular matrix components usually associated with the basal laminae lining epithelial cells.

Phosphoprotein patterns in Onchocerca volvulus developmental stages.

Living females and microfilariae of Onchocerca volvulus incubated in culture medium containing [32P]orthophosphate were observed to phosphorylate their proteins rapidly. Patterns of phosphoproteins in extracts from these labelled parasites were compared after two dimensional electrophoresis and autoradiography. Protein extracts from eggs, microfilariae and adult females of O. volvulus were phosphorylated in the presence of [gamma-32P]ATP, magnesium acetate, and added cyclic AMP-dependent protein kinase or the endogenous protein kinase present in the extracts. Patterns of phosphoproteins were compared after separation by single and two-dimensional gel electrophoresis followed by autoradiography. Common phosphopeptide bands were observed when phosphorylated extracts from adult females, microfilariae and eggs were compared. However, extracts from eggs displayed unique phosphorylated polypeptides of M(r) 30,000 and 34,000 that were absent from the extracts from microfilariae. Furthermore, two phosphorylated polypeptides of M(r) 47,000 and 76,000 were detected in extracts from microfilariae but not from eggs. These results indicate that O. volvulus parasites may phosphorylate different proteins at different stages of their development.

Characterization of the N-linked oligosaccharides in glycoproteins synthesized by microfilariae of Dirofilaria immitis.

In this report, we describe studies on the structures of the N-linked oligosaccharides contained in glycoproteins synthesized by microfilariae of the canine heartworm, Dirofilaria immitis. Microfilariae were incubated in media containing either 2-[3H]mannose, 6-[3H]glucosamine, or 6-[3H]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were radiolabeled by both the mannose and glucosamine, whereas glycoproteins were not radiolabeled by the galactose. Glycopeptides from these total glycoproteins were fractionated and purified by serial lectin affinity chromatography, and the structures of the oligosaccharides in the isolated glycopeptides were analyzed by a variety of techniques. The N-linked oligosaccharides were shown to contain mannose (Man), fucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). However, they lacked sialic acid and galactose, which are commonly found in mammalian glycoproteins. GalNAc was shown to be in an unusual terminal position and beta-linked in the sequence GalNAc beta GlcNAc beta Man-R, where R is the typical branch of complex-type N-linked oligosaccharides. Similar structures were recently found by us to be synthesized by the helminthic parasite Schistosoma mansoni. These results demonstrate that glycoproteins synthesized by microfilariae of D. immitis have unusual carbohydrate moieties and may lead to a better understanding of the specific roles of glycoprotein oligosaccharides in host-parasite interactions.

The sheath of the microfilaria of Wuchereria bancrofti has albumin and immunoglobulin on its surface.

Using direct fluorescent antibody analysis it was shown that the sheath of live microfilariae of Wuchereria bancrofti has human albumin and the immunoglobulin G subclasses IgG1 and IgG4 on its surface.

Cytochemical localization of carbohydrate residues in microfilariae of Wuchereria bancrofti and Brugia malayi.

We investigated the localization of carbohydrate residues on the surface structures of microfilariae of Wuchereria bancrofti and Brugia malayi, using a panel of 10 different gold-labeled lectins and chitinase. The sheath, a structure that encloses the microfilariae, is not a homogeneous structure, presenting two clearly distinct layers. The outer layer is more electron dense and was not labeled with the lectins. The inner layer is less dense and was intensely labeled with lectins, especially those that recognize D-galactose and N-acetyl-D-galactosamine. Small differences were observed in the lectin labeling pattern of microfilariae of W. bancrofti and B. malayi. D-galactose and fucose were observed in the cuticle of both species. Chitin, as revealed with gold-labeled chitinase, was observed in the cuticle of microfilariae of W. bancrofti but not in B. malayi.

Chemical composition of Litomosoides carinii microfilarial sheaths.

Litomosoides carinii microfilariae were exsheathed by freezing and thawing, and the sheaths were separated by filtration. Samples of pure sheaths thus obtained were hydrolyzed, methanolyzed or oxidized with nitric acid under pressure at 300 degrees C, respectively, and were analyzed for amino acids, sugars, fatty acids or for metal ions and phosphorus. Almost 75% of the sheath dry weight could thus be accounted for. Amino acids (55 weight %) were the major constituents, and amongst these glutamine and proline (approximately 11% each). The detection of 2% cysteine/cystine indicated the possible presence of disulfide crosslinks. Besides amino acids, approximately 8% of sugars--roughly equimolar amounts of (N-acetyl)galactosamine and uronic acids--1.5% of monovalent cations (Na+ and K+) and 9.5% of phosphate were detected. No appreciable amounts of fatty acids, neutral sugars, neuraminic acid, or (N-acetyl)glucosamine (i.e. no chitin) were found.

Litomosoides carinii: extraction of the microfilarial sheath components and antigenicity of the sheath fractions.

Microfilarial sheaths of Litomosoides carinii were isolated and extracted with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2ME). Extraction with SDS alone did not alter the ultrastructure of the sheaths and yielded five polypeptides (27-67 kDa) that were not recognized by antibodies of infected hosts but reacted with antibodies to host-serum proteins. 2ME treatment caused partial solubilization of the sheaths (45% as determined by amino acid analysis), which could be further improved by combining 2ME with SDS. The remainder showed filamentous/threadlike structures on electron microscopic examination. As compared with whole sheaths, the insoluble proportion was markedly enriched in alanine and cysteine but contained less galactosamine, serine, and threonine. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 2ME/SDS-extractable components showed 12-16 bands of 14- greater than 120 kDa. A predominant component had an apparent molecular mass of 22 kDa. Two bands (42 and 120 kDa) could be stained with Coomassie blue but showed "negative" staining when gels were stained with silver. Several components (but not the 22-kDa polypeptide) bore phosphocholine epitopes. Apart from the negatively staining bands, most of the 2ME-soluble sheath components were recognized by antibodies of L. carinii-infected Mastomys coucha. Except for several polypeptides that had been unspecifically recognized by IgM, the antibody response to sheath components started at the end of the prepatent period.

A major Litomosoides carinii microfilarial sheath glycoprotein (gp22): amino terminal sequence and immunological studies with corresponding synthetic peptides.

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3-6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.