Distribution of Taenia solium Diagnostic Glycoproteins in the Different Developmental Stages of the Parasite.

Taenia solium is a helminth parasite that causes 2 diseases in humans: cysticercosis and taeniasis. The establishment of T. solium metacestodes in the central nervous system causes neurocysticercosis, while development of the adult tapeworm in the small intestine causes taeniasis. Serological diagnosis of neurocysticercosis is performed by Western blot with an enriched fraction of glycoproteins that has been extensively used for clinical diagnosis and epidemiological surveys. The lectin-bound fraction that is used for this assay contains 7 antigenic glycoproteins. These antigenic proteins are considered to be highly specific for cysticercosis when tested with heterologous parasitic diseases. However, recent studies show that people with taeniasis have cross-reactive antibodies against the neurocysticercosis diagnostic glycoproteins and vice versa. Nevertheless, it is not known if these diagnostic proteins are expressed in the adult stage of the parasite. In this paper, we describe the location of 3 of these glycoproteins in T. solium adults and cysticerci using polyclonal antibodies raised against a synthetic peptide based on the amino acid sequence of TS14, a recombinant protein T24H, and the native GP50. The glycoproteins' distribution was different in invaginated and evaginated cysticerci as well as in adult tapeworms. Specifically, the 3 glycoproteins studied were differentially expressed during embryogenesis. Our findings indicate that expression of the diagnostic glycoproteins is developmentally regulated; this is noteworthy since these glycoproteins are considered specific for the diagnosis of neurocysticercosis but nevertheless are present in different structures throughout the development of T. solium. Here we describe the glycoprotein expression and localization, which can be important in understanding their biological functions. In addition, our results help clarify the cross-reaction observed between people with neurocysticercosis and taeniasis to TS14, T24H, and GP50, which are used as diagnostic antigens for neurocysticercosis.

Protein profiles of Taenia solium cysts obtained from skeletal muscles and the central nervous system of pigs: Search for tissue-specific proteins.

Taeniasis/cysticercosis caused by the tapeworm Taenia solium is a parasite disease transmitted among humans and pigs, the main intermediate host. The larvae/cysts can lodge in several tissues of the pig, i.e. skeletal muscles and different locations of the central nervous system. The molecular mechanisms associated to tissue preferences of the cysts remain poorly understood. The major public health concern about this zoonosis is due to the human infections by the larval form in the central nervous system, causing a highly pleomorphic and debilitating disease known as neurocysticercosis. This study was aimed to explore the 2DE protein maps of T. solium cysts obtained from skeletal muscles and central nervous system of naturally infected pigs. The gel images were analyzed through a combination of PDQuest™ and multivariate analysis. Results showed that differences in the protein patterns of cysts obtained from both tissues were remarkably discrete. Only 7 protein spots were found specifically associated to the skeletal muscle localization of the cysts; none was found significantly associated to the central nervous system. The use of distinct protein fractions of cysts allowed preliminary identification of several tissue-specific antigenic bands. The implications of these findings are discussed, as well as several strategies directed to achieve the complete characterization of this parasite's proteome, in order to extend our understanding of the molecular mechanisms underlying tissue localization of the cysts and to open avenues for the development of immunological tissue-specific diagnosis of the disease.

Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis.

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.

Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci.

The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.

Taenia crassiceps organic acids detected in cysticerci.

Taenia crassiceps cysticerci is used as an experimental model to cysticercosis studies; however there are subcutaneous cases of cysticercosis caused by these cysticerci. It remains unclear in the literature the energetic and fatty acid metabolism in cestodes. Its metabolic study may provide knowledge of pathways that may serve as potential anti-helminthic drugs sites of action. In this work we studied the citric acid cycle organic acids and the fatty acid oxidation in cysticerci removed from mice with 21 and 42 days of infection in two different evolutive stages: growing and final. The organic acids were extracted using perchloric acid and analyzed by HPLC methodology. We found significant statistically differences in oxalate, malate, lactate, and beta-hydroxybutirate concentrations between cysticerci. These results indicate the aerobic metabolism in vivo in spite of the low oxygen concentration of its habitat, and also indicate the presence of fatty acid oxidation as an alternative energetic source.

Biochemical characterization of annexin B1 from Cysticercus cellulosae.

Annexin B1 from Cysticercus cellulosae has recently been identified using immunological screening in an attempt to find novel antigens for vaccine development against cysticercosis. The protein possesses anticoagulant activity and carries significant therapeutic potential due to its thrombus-targeting and thrombolytic properties. We investigated the biochemical properties of annexin B1 using liposome and heparin Sepharose copelleting assays, as well as CD spectroscopy. The calcium-dependent binding to acidic phospholipid membranes is reminiscent of other mammalian annexins with a clear preference for high phosphatidylserine content. A unique property of annexin B1 is its ability to bind to liposomes with high phosphatidylserine content in the absence of calcium, which might be due to the presence of several basic residues on the convex protein surface that harbours the membrane-binding loops. Annexin B1 demonstrates lectin properties and binds to heparin Sepharose in a cooperative, calcium-dependent manner. Although this binding is reversible to a large extent, a small fraction of the protein remains bound to the glycosaminoglycan even in the presence of high concentrations of EDTA. Analogous to annexin A5, we propose a model of heparin wrapped around the protein thereby engaging in calcium-dependent and calcium-independent interactions. Although the calcium-independent heparin-binding sites identified in annexin A5 are not conserved, we hypothesize three possible sites in annexin B1. Results from CD spectroscopy and thermal denaturation indicate that, in solution, the protein binds calcium with a low affinity that leads to a slight increase in folding stability.

Non-fusion expression in Escherichia coli, purification, and characterization of a novel Ca2+- and phospholipid-binding protein annexin B1.

Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture. Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis. Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein. In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml. The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein.

Utility of the cysticercus immunoblot in a patient with an atypical solitary cerebral cysticercus granuloma.

The value of the enzyme linked immunotransfer blot (EITB) assay in avoiding an invasive diagnostic procedure in a patient with an atypical solitary cerebral cysticercus granuloma is presented.

[High level expression and purification of Cysticercosis cellulose annexin32 in Escherichia coli].

In previous work, the cDNA encoding Cysticercus cellulose annexin32 has been cloned. With PCR method, two different restriction Sites were added to each end of the cDNA respectively. Then, the cDNA was inserted into prokaryotic expression vector pJLA-503. After inducing, most foreign protein was expressed in soluble form, which was up to 35% of the total protein of the bacteria. Subsequently, the recombinant Annexin32 was purified with (NH4)2SO4 stepwise precipitation, DEAE-Sepharose FF and Sephacryl S-200 HR chromatography. The final pure protein can been shown as a single band in SDS-PAGE, and the biological activity was verified by Western blot and anticoagulation activity assay.

Eosinophil chemotactic factors from cysticercoids of Hymenolepis nana.

A comparative study of eosinophil chemotactic factors was carried out using cysticercoids and oncospheres of Hymenolepis nana. Cysticercoids showed twice the chemotactic activity for eosinophils than the oncospheres. Eosinophilia induced by oncospheres and cysticercoids observed in secondary and primary infections, respectively, were discussed from the view point of the immunobiology of this parasite.

Purification of larval Taenia solium antigens by isoelectric focusing.

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.

Identification and partial characterization of a myosin-like protein from cysticerci and adults of Taenia solium using a monoclonal antibody.

The host-parasite relationship in taeniosis due to Taenia solium is practically unknown. Monoclonal antibodies were prepared against whole extracts of adult T. solium parasites and evaluated with tapeworms recovered from experimentally infected hamsters and with cysticerci from naturally infected pigs. With one antibody, mAb 4B3, it was possible to identify, purify and partially characterize a T. solium myosin. Some findings indicate that it corresponds to conventional myosin or myosin type II such as: purification with KCl, high molecular weight, size, structure (dimeric protein with globular and long tail portions), reaction with commercial anti-myosin antibodies, distribution in muscle fibres of parasites and cross-reactivity with antibodies against paramyosin from T. solium cysticerci. The reaction of the mAb was only with taeniids and not with other parasites. Also myosin was detected in faeces of infected animals and in supernatants of parasite cultures. Its presence in biological fluids may be useful for diagnosis of infected hosts.

Characterization of a cysteine proteinase from Taenia crassiceps cysts.

Human neurocysticercosis, due to infestation of the central nervous system with Taenia cysts, is a common cause of neurologic disease in endemic areas and is being increasingly recognized in the United States. Previous studies have suggested that Taenia cysts bind host IgG via Fc-like receptors and that bound IgG is degraded by the parasite, perhaps as a source of nutrients or a means of immune evasion. We now demonstrate that IgG degradation is thiol dependent and is inhibited by the cysteine proteinase inhibitor, E-64. The cysteine proteinase activity from Taenia crassiceps cysts was purified 682-fold by acid extraction, gel filtration chromatography, and anion-exchange FPLC. The cysteine proteinase appeared as a 43 kDa band on silver-stained gels. Its isoelectric point was 5.27. The purified enzyme was inhibited by cysteine proteinase inhibitors and also by chloromethyl ketone inhibitors, but not significantly by other inhibitors of serine, aspartic, or metallo-proteinases. Substrate studies showed pronounced cleavage of Z-Phe-Arg-7-amino-4-trifluoromethylcoumarin (Z-Phe-Arg-AFC), but not of substrates with neutral or positively charged amino acids in the P2 position. Km for Z-Phe-Arg-AFC was 1.0 x 10(-7) M, Kcat 58 s-1, and Kcat/Km 5.8 x 10(8) mol-1s-1. Amino acid sequencing of the amino terminus revealed a single cysteine residue with surrounding residues suggestive suggestive of a cysteine proteinase active site. The sequence, however, did not contain the conserved active site associated with enzymes of known cysteine proteinase families. This cysteine proteinase may play an important role in the interaction of Taenia cysts and host immunoglobulin and is a potential target for antiparasitic chemotherapy.

Effective immune protection of pigs against cysticercosis.

A scolex protein antigen (SPA) was prepared from cysticerci of Taenia solium obtained from naturally infected pigs. Yorkshire pigs were vaccinated with SPA plus incomplete Freund's adjuvant (IFA) or with SPA plus Corynebacterium parvum (CP). Controls were given IFA plus phosphate-buffered saline (PBS) or CP plus PBS. All animals were given three subcutaneous injections at 20-day intervals. Ten days after the third injection, the pigs were fed with 10(4) viable eggs of T. solium. All pigs developed a delayed type hypersensitivity, and a transient eosinophilia after the first dose of vaccine. High titers of specific antibodies were detected in the sera of vaccinated animals and in infected controls. A protection level of 71.43% was recorded in animals vaccinated with SPA plus IFA and of 75.00% in those vaccinated with SPA plus CP.

Purification and ultrastructural localization of surface glycoproteins of Taenia solium (Cestoda) cysticerci.

A glycoprotein-enriched fraction was obtained by Concanavalin A-Sepharose 4B affinity chromatography from a crude extract of T. solium cysticerci. The six most prominent glycoproteins with molecular sizes of 180, 103, 96, 68, 55 and 45 kDa were purified by electro-elution from polyacrylamide gel slices. Ultrastructural localization assays using hyperimmune rabbit sera to each glycoprotein, demonstrated their presence on the tegumentary surface of the bladder wall of T. solium cysticerci. Similar studies showed that the 180 kDa glycoprotein is also present on the surface of the T. solium and T. saginata adult worms, as well as in T. saginata, T. pisiformis and T. crassiceps cysticerci. The 55 kDa glycoprotein, which is one of the most abundant on the cyst surface, was found to correspond to the heavy chain of pig IgG by Western blotting.

Comparison of cysticercus extract, cyst fluid and Taenia saginata extract for use in ELISA for serodiagnosis of neurocysticercosis.

Cysticercus cellulosae extract (CS), cyst fluid (CF), and an extract of Taenia saginata adult worm (TS) were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human neurocysticercosis in Thai patients. ELISA sensitivity was found to be 78.13%, 81.25% and 62.50%, respectively. False positivity was 6.66% with CS and 0% with other antigens. CF gave positivity with a pooled visceral gnathostomiasis serum and 3 of 10 (30%) of angiostrongyliasis sera. CS produced weakly positive ELISA with pooled opisthorchiasis and visceral gnathostomiasis sera. TS gave weak positive ELISA with a pooled opisthorchiasis serum. It was concluded that CF was the best antigen for use in ELISA for serodiagnosis of human neurocysticercosis.

Histochemistry of the racemose form of Cysticercus cellulosae.

The racemose form of brain cysticercosis arises from an intense proliferation of the bladder wall after the scolex part has degenerated. The proliferating zones are 2-3 times thicker than the remaining parts of the bladder and are characterized by a densely folded tegument and thick subtegumental and parenchymal layers. The tegument and subtegumental cells contain a large amount of acid mucosubstances with sulpho groups and hydrophilic lipids, and exhibit a high activity of alkaline and acid phosphatases. The parenchyma contains a large amount of glycogen. With the gradual aging of the bladder wall and with the first signs of autolysis, the enzymatic activity as well as the amounts of glycogen, neutral and acid mucosubstances, and proteins decrease, and the hydrophobic lipids prevail over the hydrophilic ones. The results obtained are important for the differential diagnostics of cestode larval stages in the human brain.