Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphatedependent (over ATP-dependent) enzymes. To determine the flux-control distribution of glycolysis and understand its underlying control mechanisms, a kinetic model of the pathway was constructed by using the software gepasi. The model was based on the kinetic parameters determined in the purified recombinant enzymes, and the enzyme activities, and steady-state fluxes and metabolite concentrations determined in amoebal trophozoites. The model predicted, with a high degree of accuracy, the flux and metabolite concentrations found in trophozoites, but only when the pyrophosphate concentration was held constant; at variable pyrophosphate, the model was not able to completely account for the ATP production ⁄ consumption balance, indicating the importance of the pyrophosphate homeostasis for amoebal glycolysis. Control analysis by the model revealed that hexokinase exerted the highest flux control (73%), as a result of its low cellular activity and strong AMP inhibition. 3-Phosphoglycerate mutase also exhibited significant flux control (65%) whereas the other pathway enzymes showed little or no control. The control of the ATP concentration was also mainly exerted by ATP consuming processes and 3-phosphoglycerate mutase and hexokinase (in the producing block). The model also indicated that, in order to diminish the amoebal glycolytic flux by 50%, it was required to decrease hexokinase or 3-phosphoglycerate mutase by 24% and 55%, respectively, or by 18% for both enzymes. By contrast, to attain the same reduction in flux by inhibiting the pyrophosphate-dependent enzymes pyrophosphate-phosphofructokinase and pyruvate phosphate dikinase, they should be decreased > 70%. On the basis of metabolic control analysis, steps whose inhibition would have stronger negative effects on the energy metabolism of this parasite were identified, thus becoming alternative targets for drug design.Abbreviations ADH, alcohol dehydrogenase; AK, adenylate kinase; ALDO, fructose 1,6-bisphosphate aldolase; AldDH, aldehyde dehydrogenase; ATP-PFK, ATP-dependent phosphofructokinase; DHAP, dihydroxyacetone phosphate; ENO, enolase; EtOH, ethanol; F6P, fructose 6-phosphate; F(1,6)P 2 , fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Gly3PDH, glycerol 3-phosphate dehydrogenase; HK, hexokinase; HPI, hexose 6-phosphate isomerase; HXT, hexose transporter; LDH, lactate dehydrogenase; MCA, metabolic control analysis; PGAM, 3-phosphoglycerate mutase; PGK, phosphoglycerate kinase; PGM, phosphoglucomutase; 3PGDH, 3-phosphoglycerate dehydrogenase; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PPi, pyrophosphate; PPi-PFK, pyrophosphate-dependent phosphofructokinase; PPP, pentose phosphate pathway; PFOR, pyruvate:ferredoxin oxidoreductase; PFOR-AldDH, lumped reacti...

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More than one quarter of human world's population is exposed to intestinal helminth parasites. The Taenia solium tapeworm carrier is the main risk factor in the transmission of both human neurocysticercosis and porcine cysticercosis. Sex steroids play an important role during T. solium infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Thus, we evaluated the effect of progesterone administration upon the experimental taeniosis in golden hamsters (Mesocricetus auratus). Intact female adult hamsters were randomly divided into 3 groups: progesterone-subcutaneously treated; olive oil-treated as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable T. solium cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone treatment diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. The latter exhibited high expression levels of IL-4, IL-6 and TNF-α at the duodenal mucosa, accompanied with polymorphonuclear leukocytes infiltration. These results support that progesterone protects hamsters from the T. solium adult tapeworm establishment by improving the intestinal mucosal immunity, suggesting a potential use of analogues of this hormone as novel inductors of the gut immune response against intestinal helminth infections and probably other bowel-related disorders.

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Human neurocysticercosis by Taenia solium is considered an emergent severe brain disorder in developing and developed countries. Discovery of new antiparasitic drugs has been recently aimed to restrain differentiation and establishment of the T. solium adult tapeworm, for being considered a central node in the disease propagation to both pigs and humans. Tamoxifen is an antiestrogenic drug with cysticidal action on Taenia crassiceps, a close relative of T. solium. Thus, we evaluated the effect of tamoxifen on the in vitro evagination and the in vivo establishment of T. solium. In vitro, tamoxifen inhibited evagination of T. solium cysticerci in a dose-time dependent manner. In vivo, administration of tamoxifen to hamsters decreased the intestinal establishment of the parasite by 70%, while recovered tapeworms showed an 80% reduction in length, appearing as scolices without strobilar development. Since tamoxifen did not show any significant effect on the proliferation of antigen-specific immune cells, intestinal inflammation, and expression of Th1/Th2 cytokines in spleen and duodenum, this drug could exert its antiparasite actions by having direct detrimental effects upon the adult tapeworm. These results demonstrate that tamoxifen exhibits a strong cysticidal and antitaeniasic effect on T. solium that should be further explored in humans and livestock.

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Tritiated dityrosine and isotrityrosine were detected by high performance liquid chromatography (HPLC) of acid hydrolysates of cuticular proteins from larval Ascaris suum following their 96-hr in vitro incubation in [3H]tyrosine. Sixty percent of the HPLC-recovered radiolabel was present as tyrosine, 20% as dityrosine, and 6% as isotrityrosine. Approximately 13% of radioactivity was associated with several unidentified peaks. A similar distribution of radioactivity was observed in acid hydrolysates of cuticular proteins from young adults of A. suum following 48 hr in vitro incubation with [3H]tyrosine. The 2-mercaptoethanol (2ME)-insoluble cuticular protein from the larval stages had a higher rate of synthesis of [3H]dityrosine than did the 2ME-soluble cuticular proteins, whereas the 2ME-soluble cuticular proteins had higher rates of synthesis of [3H]isotrityrosine. Pulse-chase studies of A. suum larvae demonstrated a relatively low rate of synthesis of both dityrosine and isotrityrosine. The addition to the culture media of the peroxidase inhibitors, phenylhydrazine (PHEN), 3-amino-1,2,4-triazole (AT), and N-acetyltyrosine (NAT) reduced the amount of [3H]tyrosine synthesized into both dityrosine and isotrityrosine. In a cell-free system, soluble extracts of A. suum larvae also converted radiolabeled tyrosine to dityrosine; isotrityrosine was produced by some extracts. The rate of conversion correlated with time of incubation and the volume of added extract and was inhibited by AT, NAT, and PHEN, with PHEN being the most potent inhibitor. The results of the present study suggest that the tyrosine residues of the cuticular proteins are posttranslationally modified by the formation of dityrosine and isotrityrosine cross-links. This modification is most likely mediated by a peroxidase.

A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromatographic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab')2 fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5 kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.