[Show abstract][Hide abstract] ABSTRACT: A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.
Molecular and Biochemical Parasitology 11/1999; 104(1):81-91. · 1.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14α demethylase inhibitor ketoconazole and two inhibitors of Δ24(25)-sterol methyl transferase, 20 piperidin-2-yl-5α-pregnan-3β-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3β-ol (ergosta-7-en-3β-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3β-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100–300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that Δ14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-14C]-acetate into the parasite’s endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both Δ5 and Δ22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.
Antimicrobial Agents and Chemotherapy 07/1998; 42(7):1771-7. · 4.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.
[Show abstract][Hide abstract] ABSTRACT: Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms
of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced
parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used
drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful
in the treatment of human long-term Chagas' disease, a condition that is currently incurable.
[Show abstract][Hide abstract] ABSTRACT: We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of delta 24(25) sterol methyl transferase (E.C. 126.96.36.199) of yeasts and fungi on epimastigotes and amastigotes on Trypanosoma (Schizotrypanum) cruzi, the causative agents of Chagas disease, as well as its chemotherapeutic effects in a murine model of the disease. On the epimastigote form proliferating in liver infusion tryptose medium at 28 degrees C 22,26-azasterol (AZA), a cholestanol analog with a 6-membered aza ring as a side chain produced a dose-dependent reduction of the growth rate up to 3 microM, but at 10 microM complete growth arest and cell lysis took place after 120-144 h. For 24(R,S),25-epiminolanosterol (EIL), complete growth arrest and lysis took place with 6 microM. In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14 alpha-demethylase such as ketoconazole or SDZ 89,485, as indicated by concave isobolograms and fractional inhibitory concentrations ranging from 0.11 to 0.46. Analysis of the sterol composition in control and treated cells by thin-layer and capillary gas-liquid chromatography coupled to mass spectrometry showed that growth inhibition correlated with the complete disappearance of the native endogenous sterols of the parasite (ergosterol and 24-ethyl analogs) and the accumulation of 24-desalkyl sterols. Against the clinically relevant amastigote form proliferating inside cultured Vero cells at 37 degrees C, AZA eradicated the parasite of 100 nM, while the corresponding concentration for EIL was 300 nM. Synergic effects of both inhibitors when combined with ketoconazole against this form of the parasite was demonstrated using a three-dimensional analytic method which allowed the identification of optimal drug concentrations. Finally, it was found that daily oral administration of AZA at 50 mg/kg/day for a total of 43 doses to mice infected with a lethal inoculum of T. cruzi allowed survival of all treated animals 25 days after infection, while all control (untreated) animals were dead at this point of time. Increased survival correlated with a 90% reduction in parasitemia in the treated animals. The antiparasitic effects of the azasterol were potentiated in combined treatments with ketoconazole. This is the first report of a successful application of a sterol methyl transferase inhibitor as a chemotherapeutic agent in a protozoal infection.
[Show abstract][Hide abstract] ABSTRACT: Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.
[Show abstract][Hide abstract] ABSTRACT: We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.
[Show abstract][Hide abstract] ABSTRACT: In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24-25-kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completely sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N-terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C-mediated release of GPI-anchored molecules.
Biology of the Cell 02/1992; 75(1):11-7. DOI:10.1016/0248-4900(92)90119-L · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The in vitro antiproliferative effects of ICI 195,739, a recently developed bis-triazole derivative (T. Boyle, D. J. Gilman, M. B. Gravestock, and J. M. Wardleworth, Ann. N.Y. Acad. Sci. 544:86-100, 1988; J. F. Ryley, S. McGregor, and R. G. Wilson, Ann. N.Y. Acad. Sci. 544:310-328, 1988), on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi and some aspects of its mechanism of action are described. Despite previous claims that triazole compounds act on susceptible organisms by essentially the same mechanism demonstrated for the imidazole compounds, i.e., by interfering with the synthesis of ergosterol at the level of the cytochrome P-450-dependent C-14 demethylation of lanosterol, our results indicate that ICI 195,739 acts on T. cruzi epimastigotes by a dual mechanism which involves blockade of ergosterol byosynthesis and a second, still-unidentified target whose alteration leads to immediate growth arrest. Although ICI 195,739 blocks ergosterol biosynthesis at the level of C-14 lanosterol demethylation, as shown by gas-liquid and thin-layer chromatography, growth arrest in ICI 195,739-treated cells is not related to the depletion of the endogenous ergosterol pool, contrary to what was previously found for ketoconazole, the reference compound among antifungal and antiprotozoal azole derivatives. Consistent with this observation is the fact that the concentration of ICI 195,739 required to inhibit de novo synthesis of ergosterol in epimastigotes by 50% is 60 nM, which is essentially identical to that previously found for ketoconazole under identical conditions, while the minimum concentration required to produce complete growth inhibition is 0.1 microM, which is 300 times lower than that of ketoconazole. With respect to the intracellular amastigote form proliferating inside vertebrate (Vero) cells, 10 nM is sufficient to eradicate the parasite completely in 96 h, with no effects on the host cells; this concentration is identical to that previously found for ketoconazole. Growth inhibition and morphological alterations induced by ketoconazole can be reserved by exogenously added ergosterol but not by cholesterol; for ICI 195, 739, neither sterol is capable of reserving the drug effects. Contrary to what was observed for ketoconazole, the in vitro antiproliferative effects of ICI 195, 739 on both forms of the parasite are not potentiated by the simultaneous presence of terbinafine, an allylamine which blocks ergosterol production by the parasite at a different level of the sterol biosynthetic pathway. These results, together with those of an accompanying study of the ultrastructural alterations induced by the drug, strongly support the notion that ICI 195, 739 acts on T. cruzi by a novel combination of biochemical and cellular effects, which could explain its extraordinary potency in vivo against the parasite.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the growth-inhibitory effects of two ergosterol biosynthesis inhibitors, the dioxolane imidazole ketoconazole and the allylamine SF 86-327, alone and in combination, on the proliferative stages of Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease. Proliferation of epimastigotes in liver infusion-tryptose medium at 28 degrees C was immediately arrested by any of these drugs at greater than or equal to 3 x 10(-5) M; cell lysis occurred 24 h later. Below that concentration, SF 86-327 at concentrations down to 1 x 10(-6) M stopped growth after 48 h. In contrast, ketoconazole slowed cell growth only moderately, but proliferation finally stopped and cell lysis occurred after 120 h at 3 x 10(-6) M. Synergistic effects could be observed when the two drugs were used in combination: the concentration of SF 86-327 required to reduce the cell growth to 25% of controls in 144 h was reduced 33-fold in the presence of 1 x 10(-6) M ketoconazole, which by itself reduced growth only by 30%. Amastigotes, proliferating in Vero cells at 37 degrees C, were much more susceptible to both drugs, but ketoconazole was definitely a more potent antiparasitic agent than the allylamine in this system: whereas the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 1 x 10(-7) M and that required to completely eradicate the parasite was 3 x 10(-6) M, for ketoconazole these concentrations were 1 x 10(-10) M and 1 x 10(-8) M, respectively. Again, strong synergistic effects were observed when the drugs were used in combination: the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 100-fold lower in the presence of 10(-11) M ketoconazole, which by itself had no effects on amastigote proliferation. The parasite was completely eradicated when the drugs were used in combination at concentrations as low as 10(-9) M. Synergy of the antiproliferative effects of the drugs on both froms of the parasite was further demonstrated by concave isobolograms. On the other hand, SF 86-327 at 10(-5) M had no effects on the proliferation of Vero cells, whereas ketoconazole at 10(-7) M reduced the proliferation of these cells by 50%.
[Show abstract][Hide abstract] ABSTRACT: Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
Annals of the New York Academy of Sciences 02/1988; 544(1 Antifungal Dr):357-8. DOI:10.1111/j.1749-6632.1988.tb40421.x · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that adhesion to fibroblastic cells of cell culture-derived trypomastigotes of Trypanosoma cruzi probably occurs through a ligand-receptor interaction. The results now obtained indicate that solubilization with a mild detergent (‘Chaps’, 0.8%) of 125I-surface proteins of trypomastigotes, followed by detergent removal and interaction of the solubilized proteins with a monolayer of intact Vero cells, brings about binding to the cells of a parasite surface protein, which exhibits a molecular weight of 83 000 and isoelectric point of 8.1–8.6 upon two-dimensional polyacrylamide gel electrophoresis. This polypeptide was detected in extracts of highly adherent, extracellularly incubated parasites, but not in extracts of poorly adhesive, recently released trypomastigotes. The detergent-free extracts of incubated trypomastigotes inhibit attachment of live parasites to Vero cells, while extracts of fresh trypomastigotes are nearly ineffective. Binding of the parasite polypeptide to the cells is stimulated by parasite trypsinization or activation in the presence of tunicamycin, and it is inhibited by the presence of mannan or by Vero cell trypsinization, thus showing a similar behaviour to that observed for parasite attachment to Vero cells under these conditions. These results suggest that the surface membranes of activated, highly adherent T. cruzi trypomastigotes contain an 83 kDa polypeptide which acts as a lectin-like protein that can interact with the surface of Vero fibroblasts, probably through mannose residues of a glycoprotein receptor of the host cell.
[Show abstract][Hide abstract] ABSTRACT: To assess the possible action of cAMP on the cell differentiation of Trypanosoma cruzi, we determined both cAMP levels and cAMP-binding activities of epimastigotes and trypomastigotes of this parasite. Trypomastigotes showed a 4-fold higher cAMP content and a 2.5-fold increase in the specific activity of a cAMP-binding protein with identical properties to that of epimastigotes. The high levels of cAMP present in trypomastigotes strongly suggest a role of this cyclic nucleotide on the differentiation of T. cruzi.
[Show abstract][Hide abstract] ABSTRACT: Heat-inactivated calf-, human-, and especially fetal calf serum stimulate infection of Vero cells by cell culture-derived trypomastigotes of Trypanosoma cruzi: the stimulatory effect is more marked with extracellular activated parasites or trypsinized trypomastigotes than with recently released parasites. The augmented invasion is not the consequence of a stimulation of attachment of trypomastigotes to host cells. Various sialoglycoproteins like fetuin, transferrin, fibrinogen, α-1-antitrypsin, mucin and goat-IgG are also effective in enhancing in vitro infectivity. Colominic acid also stimulates invasion, but other non-sialic polyanionic compounds are either ineffective (chondroitin sulfate, poly-aspartic acid) or inhibitory (heparin, phytic acid, myo-inositol hexasulfate). Fetuin, the best stimulatory compound tested, gives half-maximal activation with approximately 0.03 mg ml−1, and total activation with 0.5–1 mg ml−1. The enhancement of infectivity is time-dependent (2–3 h for maximal activation) at 37°C and does not occur at 0°C. Desialidated-fetuin or -fetal calf serum do not stimulate infectivity at all. Treatment with fetuin of parasites alone (or Vero cells alone), followed by removal of free fetuin and by interaction with untreated Vero cells (or parasites) indicates that the stimulation effect of fetuin occurs mainly on the trypomastigotes. No specific binding of [125I]fetuin to the parasites could be demonstrated, and incubation with exogenous neuraminidase of trypomastigotes previously activated by fetuin, reverses nearly completely the stimulation. Since a transfer of sialyl residues from exogenous sialoconjugates to epimastigotes has been recently reported [Previato et al. (1985) Mol. Biochem. Parasitol. 16, 85–96] and the behaviour of the stimulation by sialoglycoproteins of trypomastigote infectivity is compatible with such an enzymatic reaction, the hypothesis is here advanced that sialylation of T. cruzi surface components is involved in the invasion of fibroblastic cells by cell culture-derived trypomastigotes.
[Show abstract][Hide abstract] ABSTRACT: We compared the major polypeptides of epimastigotes and trypomastigotes of T. cruzi., by submitting total parasite lysates to electrophoresis in polyacrylamide gels (SDS-PAGE), protein staining with Coomassie brilliant blue, laser densitometry, or immunoblotting with sera derived from infected individuals (Chagas' disease). Epimastigotes and trypomastigotes displayed extensive homology, the differences being quantitative, except for a trypomastigote-specific band of Mr 75 000 which reacted with chagasic sera. Immunoblotting with chagasic sera confirmed the electrophoretic homology of epimastigotes and trypomastigotes. Upon antigenic dilution, a cluster of antigenic bands in the range of Mr 150 000 to 75 000 prevailed in the trypomastigotes, whereas the epimastigotes displayed more abundance of antigenic bands in the range of Mr 72 000 to 36 000.
[Show abstract][Hide abstract] ABSTRACT: It has been shown previously that the capability to adhere to and infect fibroblastic cells by Trypanosoma cruzi is expressed only partially in trypomastigotes recently liberated from infected fibroblasts, but these parasites can increase several-fold their adhesion and infectivity by a time-dependent extracellular incubation. It is now shown that polyacrylamide gel electrophoresis patterns of 125I-labelled surface proteins of the parasites change during the activation process and that protease inhibitors of diverse specificity can block both these changes and the development of adhesion and infectivity. Treatment of fresh trypomastigotes with different proteases increases immediately adhesion and infection. The effect of trypsin has been studied in detail and it was found that this protease stimulates adhesion 4- to 6-fold, even in trypomastigotes obtained and assayed in the absence of serum. Trypomastigotes incubated for various periods and then exposed to trypsin increase their adhesion to values similar to those attained by prolonged incubation of trypomastigotes alone, but infection is stimulated in fresh trypomastigotes only. Trypomastigotes whose development of activation has been inhibited either by protease inhibitors, puromycin, and tunicamycin, and are thereafter trypsinized, show respectively, that: adhesion and infection are restored immediately to the same high values obtained when untreated controls are trypsinized, adhesion is restored, but not infection, and infection is not restored. These results suggest that the adhesion step of T. cruzi trypomastigotes to fibroblastic cells depends on a membrane protein(s) that is (are) already present in an inactive or hidden form in parasites recently liberated from infected fibroblasts. Upon extracellular maturation of these trypomastigotes this proteins(s) is activated or unmasked, probably through an endogenous proteolytic process, whose expression requires protein synthesis. The penetration step requires biosynthesis of a tunicamycin-sensitive glycoprotein(s) of the parasite and its full expression necessitates serum.
[Show abstract][Hide abstract] ABSTRACT: Detailed knowledge of the mechanism by which vertebrate cells are invaded by the haemoflagellated parasite Trypanosoma cruzi is of paramount importance for understanding one of the early events in the life cycle of this obligatory intracellular parasite. The ability to infect vertebrate fibroblastic cells was found to be only partially expressed in trypomastigotes that were recently liberated from cell cultures. These trypomastigotes could increase by several fold their capability for adhesion and infection by a time-dependent in vitro process. This activation phenomenon was used to study how certain inhibitors of macromolecular biosynthesis (actinomycin D, puromycin, tunicamycin), proteases, protease inhibitors, and a combination of them, acted on adhesion and infection. The effect of exposing the parasites and, independently, the host fibroblasts to various lectins and carbohydrates was also investigated. Most of these treatments either inhibited or stimulated adhesion and infection. Exposure of fibroblastic cells to trypsin and to drugs altering the cytoskeleton impaired their susceptibility to infection. Tunicamycin blocked the recovery of infection, but not of adhesion, in trypsinized cells. The results obtained have been interpreted as indicating that the process of fibroblast infection by T. cruzi trypomastigotes occurs through two distinct and independent steps (adhesion and penetration), mediated by components of both the parasite and the host cell. The parasite components seem to be: (a) a lectin-like protein involved in adhesion; (b) an activating inducible system, probably of proteolytic nature, which enhances parasite adhesion; and (c) a tunicamycin-sensitive glycoprotein, related to penetration only. As for the fibroblastic cell components, these are postulated to be two glycoproteins (one insensitive and the other sensitive to tunicamycin), which are involved in the steps of parasite attachment and penetration, respectively.