Javier R. Ambrosio

Detection of genetic variation in Taenia solium

Genetic variability among Taenia solium isolates was studied in 160 cysticerci from 6 pigs, 4 from Mexico, 1 from Honduras, and 1 from Tanzania. Random amplified polymorphic DNA (RAPD) analysis performed with 4 commercial primers showed 88% polymorphic loci and an average heterozygosity of 0.077; however, several alleles were fixed within each isolate. Linkage disequilibrium analysis indicated that 3 of the 6 isolates had a random association of alleles, whereas the other 3 had a clonal structure. These results suggest the existence of local lineages in T. solium, with events of genetic recombination within them.

Observations on the musculature and isolated muscle fibres of the liver fluke, Fasciola hepatica

The liver fluke, Fasciola hepatica relies on a well-developed muscular system, not only for attachment, but for many aspects of its biology. Despite this, little is known about the system beyond the gross organization of the main somatic muscle layers. In the present study, a range of techniques have been applied to F. hepatica in order to understand more about various aspects of muscle organization, biochemistry (in terms of muscle proteins) and identity of isolated muscle fibres. Scanning electron microscopy has provided a direct visualization in situ of the somatic muscle layers and the organization of the muscle fibres within the ventral sucker. The muscle bundles contributing to the main somatic muscle layers are made up of up to 10 individual muscle fibres. Phalloidin staining for actin, in conjunction with confocal microscopy, confirmed the presence of 2 main somatic muscle layers (outer circular, inner longitudinal), beneath which lies a third layer of oblique muscle fibres. The use of propidium iodide in combination with phalloidin staining for actin demonstrated that the cell bodies associated with the 2 main somatic muscle layers are situated beneath the longitudinal muscle layer and are connected to their respective muscle fibres by short cytoplasmic processes. Myosin immunoreactivity was demonstrated in the somatic muscle layers and in the muscle layers surrounding various organ systems within the fluke. Double labelling for actin and myosin confirmed the co-localization of the 2 muscle proteins in the muscle fibres of the ventral sucker. Muscle fibres from the somatic muscle layers and the ventral sucker have been isolated and images obtained with phase-contrast microscopy and scanning electron microscopy. The muscle fibres contain actin and myosin, but lack a nucleus, the connection with the cell body having been broken during the isolation procedure.

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T.cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

Actin expression in Taenia solium cysticerci (cestoda): tisular distribution and detection of isoforms

Identification, localization and partial biochemical characterization of actins expressed in the larval stage of the cestode parasite Taenia solium has been carried out. Frozen tissue sections of cysticerci, the larval stage of this parasite, were reacted with rhodamine‐phalloidin, parasite actin was purified by polymerization in the presence of K+, mg++and ATP actin was analyzed by SDS—PAGE and two‐dimensional gel electrophoresis, and immunoblotting of actin was performed in PVDF membranes and with commercial anti‐actin monoclonal antibodies. Parasitic tissues showed different fibrous actin fluorescence patterns, which correlated with the expression of isoactins. Purified globular actin had a similar molecular mass to rabbit commercial actin (∼44 kDa). Actin was resolved into seven isoforms, indicating a family of actin genes.

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross‐reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin—myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.

Visualization and 3D Reconstruction of Flame Cells of Taenia solium (Cestoda)

Background Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. Methodology/Principal Findings Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. Conclusions/Significance We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

Analysis of the effect of a 2-(trifluoromethyl)-1H-benzimidazole derivative on Trichinella spiralis muscle larvae.

Albendazole and mebendazole are widely used in the treatment of trichinellosis; however, chemotherapy failure has been reported. In an effort to develop new anthelminthic compounds, we examined a previously synthesized 2-(trifluoromethyl)-1H-benzimidazole derivative (1) that showed good in vitro activity against Trichinella spiralis muscle larvae but low in vivo efficacy. In order to improve the solubility of compound 1, an inclusion complex with 2-hydroxypropyl-β-cyclodextrin (1/HP-βCD) was prepared. When 1/HP-βCD was tested in vivo, it significantly reduced the ML burden (84%). In addition, a proteomic analysis of T. spiralis ML treated with 1 revealed significant changes in the expression levels of proteins involved in energy metabolism and the cytoskeleton of the parasite. Compound (1) also induced extensive ultrastructural changes in the cuticle, hypodermis and midgut of the parasite.

Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

Immunodiagnosis of porcine cysticercosis: Identification of candidate antigens through immunoproteomics

Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.