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Characterization of the gene family encoding a host-protective antigen of the tapeworm Taenia ovis.

Genomic structure has been determined for a gene encoding a host-protective antigen of the parasitic platyhelminth Taenia ovis. An incomplete cDNA, known as 45W, encodes the protective antigen. Southern hybridisation experiments using 45W cDNA as a probe, revealed that the 45W gene was a member of a multigene family. Differential Southern hybridisation and rapid amplification of cDNA end (RACE) experiments were used to characterise the related genes, allowing the full-length coding region of the 45W encoded antigen to be determined. The gene family comprises a minimum of four members per haploid genome with each member showing varying degrees of 5' and 3' homology with respect to the 45W cDNA. A close homologue of the 45W gene, designated 45S, differed from 45W at 11 of 985 nt comprising the full-length mRNA. Sequencing of several independent RACE products for both 45W and 45S identified a cDNA which may be a product of homologous recombination between these genes, suggesting that the two genes may be alleles. Homologous recombination in genes which encode a host protective antigen such as 45W would provide a mechanism by which antigenic variants could arise.

Amino Acid Sequence↗

Phloretin inhibition of glucose transport by the tapeworm Hymenolepis diminuta: a kinetic analysis.

Phloretin non-competitively inhibits glucose and methionine absorption by Hymenolepis diminuta (Ki = 0.24 and 1.48 mM, respectively). Inhibition of glucose transport by phloretin is reversible. Phloretin and phlorizin binding sites on the surface of H. diminuta are distinct. Phloretin does not inhibit Na+ transport independent of glucose transport in H. diminuta. The data suggest that phloretin inhibits absorptive functions across the surface of H. diminuta via non-specific binding sites associated with the lipid portion of the worm outer membrane.

Animals↗

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta.

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.

Adsorption↗

Galactose utilization by the rat tapeworm, Hymenolepis diminuta.

1. Hymenolepis diminuta incorporated label from 14C-galactose into glycogen, but the sugar would not support net glycogen synthesis. Glucose stimulated the incorporation of label from 14C-galactose into glycogen, while glycerol did not. 2. During incubations in galactose, large internal pools of galactose and galactose 1-P accumulated, while the concentration of glucose 6-phosphate remained unchanged. 3. In vitro culture experiments indicated that galactose would not support worm growth. Therefore, while galactose can be metabolized to a limited extent, it cannot substitute for glucose as a nutrient source.

Animals↗

Glycogen synthase in the rat tapeworm, Hymenolepis diminuta--I. Enzyme activity during development and with crowding.

1. Activity of glycogen synthase (E.C. 2.4.1.11) in Hymenolepis diminuta (Cestoda: Cyclophyllidea) was investigated as a function of development and with crowding. 2. Synthase activity was low in the anterior and posterior ends of the worms and highest in the pregravid proglottids in the mid-portion of the strobila. 3. The enzyme activity increased during development of the cestode at least up to 15 days postinfection, but the increase in activity apparently was not due to conversion of the inactive to the active form. 4. Mature oncospheres also contained glycogen synthase, but the activity was lower than in strobilar tissues. 5. Synthase I activities and the proportion of total activity in the I form were generally higher in worms from high density (100 worm) infections than in those from low density (10 worm) infections.

Animals↗

Enzymes of galactose utilization in the rat tapeworm, Hymenolepis diminuta.

1. Crude enzyme preparations from Hymenolepis diminuta contained galactokinase, galactose 1-phosphate uridyl transferase and UDPgalactose 4-epimerase activity, although their specific activities were low. 2. Galactose 1-phosphate non-competitively inhibited galactose phosphorylation. This inhibition, together with the low specific activities of the enzymes in the pathway of galactose utilization, probably accounts for the inadequacy of galactose as a main nutritive carbohydrate for development of the worm.

Animals↗

Glycogen synthase in the rat tapeworm, Hymenolepis diminuta--II. Control of enzyme activity by glucose and glycogen.

1. The proportion of activity in the physiologically active I form of glycogen synthase in Hymenolepis diminuta (Cestoda) decreased in the worm when the rat host was fasted and was greatly increased in the cestode 1 hr after a 24 hr fasted rat was refed. 2. The increase in glycogen synthase I activity was due to glucose present in the host gut after feeding, not to other physiological changes in the rat intestine due to meal consumption. 3. Incubation of intact H. diminuta in vitro with glucose also resulted in the conversion of glycogen synthase D to I. 4. Glucose does not appear to affect the glycogen synthase complex directly, because neither the total synthase converted to I nor the rate of conversion was affected by glucose in a partially purified homogenate. 5. High concentrations of glycogen inhibited the synthase D to I conversion and high mol. wt glycogen was a more effective inhibitor than low mol. wt glycogen.

Adenosine Triphosphate↗

In vitro poly-(ADP-ribosyl)ation of chromatin proteins in the rat tapeworm, Hymenolepis diminuta.

1. (ADP-ribose)-transferase activity in crude chromatin of H. diminuta was demonstrated. 2. Chromatin proteins were ADP-ribosylated in vitro and selectively extracted. 60, 12 and 18% of the (ADP-ribose)n of chromatin proteins was associated with total histones, histone H1 and histone H2B, respectively. 3. The extent of oligo-(ADP-ribose) compared to total (ADP-ribose)n in the chromatin fraction, in the histone fraction, the histone H1 fraction and the histone H2B fraction was 45, 60, 26 and 49%, with an average chain length of 2.8, 2.1, 1.8 and 2.6, respectively. 4. Analysis of (ADP-ribosyl)n-ated proteins by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that histone H1, histone H2B and a 35 kDa non-histone protein were major (ADP-ribose)n acceptors.

Adenosine Diphosphate Ribose↗

Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis.

Receptor tyrosine kinases (RTKs) are crucially involved in the development of metazoan organisms and possible mediators of cell-cell communication that occurs between eukaryotic parasites and hosts. We have now cloned and characterized the complete complementary DNA (cDNA) molecule encoding a novel receptor tyrosine kinase, EmER, of the human parasite Echinococcus multilocularis. EmER shared significant amino acid sequence homologies with members of the epidermal growth factor (EGF) receptor family of different phylogenetic origin, exhibited a domain structure which is typical for this group of membrane receptors and contained all catalytically important residues at the corresponding positions. Highest homologies were detected between EmER and a previously identified receptor kinase, SER, from the parasitic trematode Schistosoma mansoni. The EmER encoding gene, emer, spans a chromosomal region of 39 kb and is composed of 23 exons. Structural comparisons indicated that emer and the EGF receptor encoding genes from mammals derive from a common ancestor. DNA/DNA hybridization experiments demonstrated that emer is present as a single copy locus in the parasite. Transcriptional analyses on in vitro cultivated parasite larvae revealed that emer is expressed in both the metacestode and the protoscolex stage, although about 10-fold higher transcription levels were detectable for the protoscolex. Northern blot experiments further indicated that emer is expressed as a single 5.2-kb transcript in parasitic larvae during an infection of the intermediate host. These results suggest an involvement of EmER in echinococcal differentiation processes towards the protoscolex stage during natural infections and provide, for the first time, structural information on an epidermal growth factor receptor-like kinase from a cestode.

Amino Acid Sequence↗

An unusual blood sequestering tapeworm (Sanguilevator yearsleyi n. gen., n. sp.) from Borneo with description of Cathetocephalus resendezi n. sp. from Mexico and molecular support for the recognition of the order Cathetocephalidea (Platyhelminthes: Eucestoda).

Sanguilevator yearsleyi n. gen., n. sp. and Cathetocephalus resendezi n. sp. are described from the Broadfin shark, Lamiopsis temmincki, in Malaysian Borneo and Carcharhinus leucas in Mexico, respectively. The new genus is unusual in its possession of internal chambers and channels in its scolex that appear to house extensive quantities of host white and red blood cells, respectively. Histology reveals an extremely intimate association between host tissue and the surface of the apical pad of the scolex. Positive staining with periodic acid-Schiff of the surface of the pad of the scolex and the linings of the chambers and channels suggests that an adhesive substance may be produced in these regions. However, explanations for how and why host blood cells come to reside within the scolex remain elusive. Cathetocephalus resendezi n. sp. is distinctive in the form of the papillae in the papillate band of the scolex and also in the inconspicuous nature of the rugose base of the scolex. Scanning electron microscopy of both new taxa as well as Cathetocephalus thatcheri, Cathetocephalus australis and an undescribed species of Cathetocephalus collected from Carcharhinus amboinensis in Australia, suggests that the papillae surrounding the pad of the scolex are of significant taxonomic utility in distinguishing among species in these groups. Parsimony and Bayesian analyses of sequence data (766 bases of 18S rDNA and 405 bases of 28S rDNA) generated from ethanol preserved specimens of C. thatcheri and S. yearsleyi, when compared with equivalent data available for 40 cestode species in GenBank, resulted in trees that support previous propositions that Cathetocephalus should be placed in the order Cathetocephalidea. The results suggest that Sanguilevator should also be considered to belong to this order.

Animals↗

Characterization of the immuno-regulatory response to the tapeworm Hymenolepis diminuta in the non-permissive mouse host.

UNLABELLED: Hymenolepis diminuta is spontaneously expelled from mice; concomitant with worm expulsion was protection against colitis induced by dinitrobenzene sulphonic acid (DNBS). Here we examined the immune response mobilized by Balb/c and C57Bl/6 male mice in response to H. diminuta and assessed the requirement for CD4+ cells (predominantly T cells) in worm expulsion and the anti-colitic effect. Wild-type (CD4+) or CD4 knock-out (CD4-/-) mice received five H. diminuta cysticercoids and segments of jejunum and mesenteric lymph nodes (MLNs), or spleen, were excised 5, 8 and 1l days later for mRNA analysis and cytokine production, respectively. In separate experiments uninfected and infected mice received DNBS by intra-rectal infusion and indices of inflammation were assessed 3 days later (i.e. 11 days p.i.). Infection of Balb/c mice resulted in a time-dependent increase in intestinal mRNA for Foxp3, a marker of natural regulatory T cells, and markers of alternatively activated macrophages (arginase-1, FIZZ1), while concanavalin-A activation of MLN cells revealed a significant increase in T helper 2 (TH2) type cytokines: IL-4, -5, -9, -10, -13. MLN cells showed a reduced ability to induce Foxp3 expression upon stimulation. CD4-/- mice did not display this response to infection, but surprisingly did expel H. diminuta. Moreover, DNBS-induced colitis in CD4-/- mice (wasting, tissue damage, elevated myeloperoxidase) was not reduced by H. diminuta infection, whereas time-matched infected CD4+ C57Bl/6 mice had significantly less DNBS-induced inflammation. IN CONCLUSION: (i) in addition to stereotypical TH2 events, H. diminuta-infected Balb/c mice develop a local immuno-regulatory response; and (ii) CD4+ cells are not essential for H. diminuta expulsion from mice but are critical in mediating the anti-colitic effect that accompanies infection in this model.

Animals↗

Molecular cloning and characterization of Ras- and Raf-homologues from the fox-tapeworm Echinococcus multilocularis.

To better understand growth regulation in the human parasitic cestode Echinococcus multilocularis, we have cloned and characterized the parasite's orthologues of the key regulatory factors Ras and Raf. Using a degenerative PCR approach a gene, emras, was identified whose gene product, EmRas, showed high homology (79% identical residues) to human Ras and contained all amino acid residues which are characteristic for this subfamily of small GTPases at the corresponding positions. Recombinantly expressed EmRas bound GTP and was farnesylated, but not geranyl-geranylated, by Echinococcus lysate in an in vitro prenylation assay. Furthermore, upon expression in yeast, emras was able to functionally complement the Saccharomyces orthologue RAS2 in an invasive growth assay. In Western blot analyses using an anti-EmRas antibody, the Echinococcus factor could be detected in lysates of the larval stages metacestode and protoscolex. By immune-histochemistry, EmRas was shown to localize to the germinal layer of the metacestode and to tegumental structures of the protoscolex, particularly around the rostellum and the sucker regions. In addition, we fully characterized the gene emraf whose product, EmRaf, displayed considerable homology to mammalian Raf-kinases and orthologous factors from Drosophila and Caenorhabditis elegans. emraf was co-expressed with emras in the larval stages metacestode and protoscolex during in vitro cultivation and during an infection of the intermediate host as assessed by RT-PCR experiments. The emraf gene was composed of nine exons and eight introns and shared four highly conserved exon-intron boundaries with the human gene encoding Raf-1, suggesting that both genes derived from a common evolutionary ancestor. Southern blot hybridizations demonstrated that emraf is a single copy gene. Using the yeast two-hybrid system, EmRaf was shown to interact with EmRas, but not with EmRal, a previously characterized orthologue of mammalian Ral GTPases. This is the first characterization of a Ras orthologue from a cestode and the first report on a Raf-like kinase from a platyhelminth. The data presented herein will form a solid basis for further investigations on Echinococcus signaling systems that are involved in growth control and development of the parasite.

Amino Acid Sequence↗

Human tapeworms in north Vietnam.

Sixty-five Taenia samples were collected from patients in a referral hospital in Hanoi, north Vietnam, for species identification by morphological and molecular techniques. PCR-RFLP of a mitochondrial 12S rDNA fragment, developed for this study, allowed direct differentiation between all Taenia spp., overcoming the disadvantages of classical morphological examination, which failed on disintegrated samples. Taenia saginata asiatica was the most common species (55.4%) followed by T. saginata (38.5%) and T. solium (6.2%). This report demonstrates the complexity of the epidemiology of Taenia spp. in Vietnam and the need for further work to reveal transmission patterns of these species.

Animals↗

Lactic acid, pyruvic acid and lactate/pyruvate ratio in the Anoplocephalid tapeworm Stilesia globipunctata infecting sheep (Ovis aries).

Lactic acid content was found to vary among the different proglottid types of the parasite. This higher amount of lactic acid might be considered as the end product of anaerobic metabolism. The pyruvic acid level of the parasitic proglottides remained lower than lactate level. The intermediary carbohydrate metabolites, viz. lactate, pyruvate and the lactate/pyruvate ratio being indicators of high rate metabolism, their levels may notably infer the metabolic status of the parasite and also the interaction between the parasite species Stilesia globipunctata and the sheep host.

Anaerobiosis↗