Lucía Jiménez

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Cloning, production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.

Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium

Abstract. A Taenia solium larval glutathione S-transferase fraction (SGSTF), composed of two proteins with Mr 25,500 (SGSTM1) and 26,500 (SGSTM2), was purified by GSH-sepharose. Its N-terminal sequence analysis revealed that both proteins are related to mammalian mu-class GST enzymes. A cDNA clone coding for SGSTM1 was isolated and the amino acid sequence analysis showed close identity with two Echinococcus GSTs and also high identity with several mu-class GSTs that have been reported. In addition, SGSTM1 presents a similar structure to mu-class GSTs, including the mu loop. The recombinant SGSTM1 is a dimeric protein with enzymatic properties clearly related to mammalian mu-class GSTs. Western blot studies indicated that SGSTM1 is not antigenically related to SGSTM2 or mammalian GSTs from rabbit, pig and rat livers. Immunization with SGSTF and SGSTM2 was highly effective in reducing cysticerci load in murine cysticercosis. In contrast, no protection was obtained using native SGSTM1 and recombinant SGSTM1 as immunogens.

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium ☆

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.

MOLECULAR CLONING AND CHARACTERIZATION OF A 2-CYS PEROXIREDOXIN FROM TAENIA SOLIUM

A Taenia solium 2-Cys peroxiredoxin (Ts2-CysPrx) clone was isolated from a T. solium adult cDNA library. The clone encodes a polypeptide comprising 197 amino acids with a predictive Mr = 21,836. It has the 2 classical cysteine domains from the typical 2-Cys peroxiredoxins, and its primary amino acid sequence shows higher identity with 2 Echinococcus 2-Cys peroxiredoxins. Northern and Southern blot hybridizations exhibit an mRNA with a size of ∼1.0 kb, encoded by 1 gene. Ts2-CysPrx was expressed in Escherichia coli and purified by anion-exchange chromatography. Biochemical analysis showed Ts2-CysPrx is a dimer composed by monomers of ∼22 kDa that presented activity with hydrogen peroxide (H2O2) and cumene hydroperoxide. It presented the catalytic mechanism for a typical 2-CysPrx because the homodimeric oxidized form is reduced to a monomeric form by thioredoxin (Trx) and by dithiothreitol (DTT) and was converted to a homodimeric oxidized form by H2O2. Western blot studies using antibodies against Ts2-CysPrx revealed that the protein is expressed during the entire T. solium life cycle, as in other Taenia species. Immunohistochemical studies indicated that Ts2-CysPrx is localized on the tegument and in tegumentary and muscle cells of cysticerci. We also show that T. crassiceps cysticerci can tolerate H2O2 levels of 2.5 mM for 2.5 hr.

A COMPARATIVE STUDY OF BIOCHEMICAL AND IMMUNOLOGICAL PROPERTIES OF TRIOSEPHOSPHATE ISOMERASE FROM TAENIA SOLIUM AND SUS SCROFA

We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa. Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a Km value of 0.758, and S. scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a Km value of 0.51. The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes. Anti–T. solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S. scrofa, human, or protozoan TPIs. These antibodies inhibited T. solium TPI activity but did not affect S. scrofa enzymatic activity. Immunizations with 1 μg of the T. solium TPI reduced 52% of cysticerci in a mouse–Taenia crassiceps model 1 mo after challenge. Our findings show that T. solium and S. scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti–T. solium TPI antibodies did not recognize S. scrofa TPI. Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.

Antimicrobial peptides (Temporin A and Iseganan IB-367): Effect on the cysticerci of Taenia crassiceps

Taenia solium infections continue being a health problem in undeveloped countries, and few effective control measures against this parasite are being applied. Antimicrobial peptides (AMPs) belong to the innate immune response and capable of destroying pathogens. We tested the ability of two AMPs, Temporin A (TA) and Iseganan IB-367 (IB-367) to damage T. crassiceps cysticerci in vitro. Doses of 200 and 400 microg/ml of TA and IB-367 caused cysticerci to shrink, lose motility, the formation of macrovesicles in the tegument, as well as decreased evagination properties. These changes were observed as early as 3-6h and became more pronounced over 24h, when the morphological changes of the bladders became evident by both light and electron microscopy. Electron micrographs of cysticerci exposed to peptides showed initial changes as collapsed microvesicles in the tegument, with formation of large vesicles and extrusion of tegumentary tissues into the surrounding media, which led to complete loss of the tegument as well as shrinkage and complete loss of structure of parenchymal tissue after 24h. Peptides administered to cysticercotic mice one month post-infection in a single intraperitoneal dose of 200 or 400 microg, reduced the parasite load by 25% for IB-367, and 50% for TA. The humoral response of infected mice does not appear capable of killing surviving cysticerci. Our studies show that in vitro, AMPs severely damage the tegument and the scolex, and open a new pathway for biological drug design or the development of transgenic animals that over express these peptides capable of killing the cysticerci in vivo.

The Hamster Model for Identification of Specific Antigens of Taenia solium Tapeworms

Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm) and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs) and excretion-secretion antigens (E/S Ag) obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis.

Use of Animals During the Mid-Archaic and the Initial Period in Pernil Alto: A Site in the Palpa Valleys, Southern Coast of Peru

Archaeological research in the Palpa valleys, southern coast of Peru, has revealed the existence of a long cultural process, during which the site of Pernil Alto had an important role in the process of settlement in the Palpa valleys during the Mid-Archaic Period (3600–3000 BC) and the Initial Period (1500–800 BC). In this paper, we present the preliminary results of archaeozoological studies of animal bones excavated in Pernil Alto. We identify the animal taxa that the Pernil Alto human group had access to and how they used them over time during the Mid-Archaic Period and the Initial Period. The description and methodological analysis of each category used to classify 1361 bone fragments is discussed. The sample studied indicates that during the Mid-Archaic Period, animal use was varied, while during the Initial Period the primary emphasis was on the use of camelids (Camelidae), with continued use of other protein sources including guinea pigs (Caviidae) and deer (Cervidae), among others.

Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product.

Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts.