Trypanosoma cruzi: Inhibition of host cell uptake of infective bloodstream forms by alpha-2-macroglobulin

The infection of murine macrophages and fibroblasts by recently isolated infective bloodstream trypomastigotes ofTrypanosoma cruzi is inhibited by the addition of human plasma protease inhibitor alpha-2-macroglobulin (₂M) or of soybean trypsin inhibitor. The ingestion of the non-infective epimastigotes by macrophages is not affected by the physiological protease inhibitor. Incubation of bloodstream trypomastigotes for 20 h in a serum-free axenic medium enhances their ability to infect macrophages in a process influenced by the temperature and sensitive to ₂M. After this period the infectivity of the parasites to cells was not sensitive to ₂M. These observations suggest that proteases located on the surface and/or secreted by the bloodstream trypomastigote form ofT. cruzi may modulate its ability to infect host cells.     

Interaction ofTrypanosoma cruzi with macrophages: effect of previous incubation of the parasites or the host cells with lectins

The effect of incubation with lectins of the macrophages or two evolutive stages ofTrypanosoma cruzi (noninfective epimastigotes and infective trypomastigotes) on the ingestion of the parasites by mouse peritoneal macrophages was studied. Lectins which bind to residues of mannose (Lens culinaris, LCA),n-acetyl-d-glucosamine orn-acetylneur-aminic acid (Triticum vulgaris, WGA), -d-galactose (Ricinus communis, RCA),n-acetyl-d-galactosamine (Phaseolus vulgaris, PHA;Dolichos biflorus, DBA; andWistaria floribunda, WFA), fucose (Lotus tetragonolobus, LTA), andn-acetylneuraminic acid (Limulus polyphemus, LPA) were used. By lectin blockage we concluded that, -d-mannose-like, -d-galactose andn-acetyl-d-galactosamine (PHA, reagent) residues, located on the macrophage's surface are required for both epi- and trypomastigote uptake, whilen-acetylneuraminic acid and fucose residues, impede trypomastigote ingestion but do not interfere with epimastigote interiorization. Macrophages'n-acetyl-d-glucosamine residues are required for epimastigote uptake. On the other hand, from theT. cruzi surface, mannose residues prevent ingestion of epi- and trypomastigotes. Galactose residues participate in endocytosis of trypomastigotes, but hinder epimastigote interiorization. Exposedn-acetyl-d-glucosamine residues are required for uptake of the two evolutive forms.n-acetylneuraminic acid residues on the trypomastigote membrane prevent their endocytosis by macrophages. These results together with those reported previously showing the effect of monosaccharides on theT. cruzi-macrophage interaction, indicate that (a) sugar residues located on the parasite and on macrophage surface play some role in the process of recognition ofT. cruzi, (b) different macrophage carbohydrate-containing receptors are involved in the recognition of epimastigotes and trypomastigotes forms ofT. cruzi, (c)n-acetylneuraminic acid residues located on the surface of trypomastigotes or macrophages impede the interaction of the parasite with these host cells, and suggest that (d) sugar-binding proteins located on the macrophage surface participate in the recognition of -d-galactose andn-acetyl-d-galactosamine residues located on the surface of trypomastigotes and exposed after blockage or splitting off ofn-acetylneuraminic acid residues. Some lectins which bind to macrophages and block the ingestion of parasites did not interfere with their adhesion.     

Development ofTrypanosoma fallisi in the leech,Desserobdella picta,in toads (Bufo americanus), and in vitro

Summary The development ofTrypanosoma fallisi ofBufo americanus from Algonquin Park, Ontario was studied by light and electron microscopy in blood culture, in its leech vectorDesserobdella (=Batracobdella) picta, and in its toad host. In culture, bloodstream trypomastigotes transformed within one day to elongate epimastigotes which divided into rosettes. These gave rise to amastigotes, spheromastigotes, stumpy and elongate epimastigotes, and slender metacyclic trypomastigotes over a 4- to 6-day period. Development in the leech crop was similar to that in culture, with fewer amastigotes and no spheromastigotes observed. The stages in the leech were similar in size to their culture counterparts, except for metacyclic trypomastigotes, which were larger in culture. Culture and leech stages possessed a well developed cytostome-cytopharyngeal complex and prominent reservosomes. The kinetoplast of crop stages was small with a rectangular profile, but became larger and basket-like in the proboscis forms. Migration of trypanosomes to the proboscis appeared to depend on the rate of digestion of the bloodmeal. Flagellates in the leech were also characterized by the presence of intracellular microorganisms. Development of culture forms to mature stages in the toad was completed within 8 days postinoculation, with the organisms transforming into the typical C-shape with a large square kinetoplast. Natural infection ofB. americanus was detected at 3 days postfeeding byD. picta and the resulting blood-stream trypomastigotes developed more slowly than inoculated culture stages.     

Characterization of the mechanism of protein glycosylation and the structure of glycoconjugates in tissue culture trypomastigotes and intracellular amastigotes of Trypanosoma cruzi

Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man7GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.     

Further characterization ofTrypanosoma cruzi GP57/51 as the major antigen expressed by differentiating epimastigotes

Study ofTrypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45–50 kDa is markedly increased on adhesion of epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a majorT. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45–50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during epimastigote differentiation suggests an improtant role for this proteinase during the life cycle ofT. cruzi.Abbreviations EDTA ethylenediaminetertraacetic acid - TLCK N--tosyl-l-lysine chloromethylketone - PMSF phenylmethylsulfonyl fluoride - NP-40 Nonidet P-40 - SDS sodium dodecyl sulfate - PBS phosphate-buffered saline - Anti-Adh rabbit antiserum against adherent epimastigotes - mAb monoclonal antibody - Ag antigen - RIA radioimmunoassay     

Ultrastructural analysis of edelfosine-treated trypomastigotes and amastigotes of Trypanosoma cruzi

We have previously reported that epimastigote forms of Trypanosoma cruzi treated with the lysophospholipid analogues (LPAs) edelfosine (ET-18), ilmofosine, and miltefosine suffered alterations in plasma membrane, mitochondrion, and lipid synthesis. In this work, ET-18 induced membrane damage in trypomastigotes and amastigotes. Incubation of epimastigotes and trypomastigotes with the three LPAs led to membrane permeabilization, which was abolished by serum addition. Treatment for 24 h in culture medium interfered the with mitochondrial status of epimastigotes, with no effect in trypomastigotes, in agreement with ultrastructural data. LPAs induced alterations in the plasma membrane of the three forms of T. cruzi and in the mitochondria of epimastigotes, suggesting that these organelles are potential targets of these analogues.     

Stable Transfection of Trypanosoma cruzi Epimastigotes with the Trypomastigote-Specific Complement Regulatory Protein cDNA Confers Complement Resistance

Trypanosoma cruzi blood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with a T. cruzi expression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene of T. cruzi trypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor of T. cruzi.     

Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi

Monoclonal antibodies were raised against the surface of trypomastigote forms of Trypanosoma cruzi. Although some of these antibodies reacted against antigens shared by trypomastigote and epimastigote or amastigote forms, the majority were trypomastigote-specific. Trypomastigote-specific monoclonal antibodies recognized all infective stages, including trypomastigotes from the bloodstream of infected mice, insect feces, tissue culture and those resulting from differentiation of epimastigotes in axenic culture media. The monoclonal antibodies H1A10 and 6A2, as well as Fab fragments from H1A10, partially prevented T. cruzi invasion of LLC-MK2 cell monolayers (inhibition of 50-70%) when present throughout the entire experiment. Both antibodies recognized an 85 kDa glycoprotein (Tc-85) of the trypomastigote surface which contains N-acetyl-D-glucosamine and/or sialic acid.     

Ultrastructural alterations and growth inhibition of Trypanosoma cruzi and Leishmania major induced by Bothrops jararaca venom

Snake venom can affect the growth of Trypanosoma cruzi and Leishmania spp. As new classes of therapeutic drugs against protozoan parasites could be derived from snake venom, alterations in the ultrastructure and growth of the epimastigotes, trypomastigotes and amastigotes of T. cruzi, as well as the promastigotes of Leishmania major, were analyzed after treatment with crude venom from Bothrops jararaca. Parasite growth (epimastigotes and promastigotes) of venom treated cultures showed a negative correlation between cell growth and venom concentration. No growth occurred at a dose of 100 microg/ml of venom, while 50% growth inhibition was obtained in the range 0.1-0.3 microg/ml. Ultrastructural observations of treated bloodstream trypomastigotes, intracellular amastigotes, as well as axenic cultures of epimastigotes and promastigotes, demonstrated mitochondrial swelling and kinetoplast disorganization. Our data show that B. jararaca venom effectively inhibited the growth of T. cruziand L. major parasites. Growth inhibition was probably related to mitochondrial impairment.     

Proton and sodium pumps regulate the plasma membrane potential of different stages of Trypanosoma cruzi

We studied the plasma membrane potential (DeltaPsi) in different stages of Trypanosoma cruzi using the potentiometric fluorescent dye bisoxonol. Our results clearly demonstrate that a proton pump plays a significant role in the regulation of DeltaPsi in all stages of T. cruzi as evidenced by depolarization of the DeltaPsi by H(+)-ATPase inhibitors dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A(1). The H(+)-ATPase appeared to be activated by acidic conditions in trypomastigotes as evidenced by hyperpolarization of the DeltaPsi upon addition of acid. In contrast to epimastigotes and amastigotes, the DeltaPsi of trypomastigotes was markedly sensitive to extracellular Na(+) and K(+) concentrations and evidence was provided for an outward directed Cl(minus sign) channel in this stage as well. Additionally, for the first time, the existence of an ouabain-sensitive functional sodium pump was demonstrated in T. cruzi based on the depolarization of the DeltaPsi in trypomastigotes by ouabain in the presence of Na(+). In the epimastigotes, ouabain had no effect on the DeltaPsi in a sodium rich buffer. However, ouabain induced an additional significant depolarization in these stages when their DeltaPsi was already partially depolarized by the H(+)-ATPase inhibitor dicyclohexylcarbo-diimide, supporting the presence of an ouabain-sensitive sodium pump whose activity is masked by the H(+)-ATPase. In the amastigotes no evidence for a functional sodium pump could be found. In support of an inwardly directed K(+) channel, the DeltaPsi was hyperpolarized by K(+) free buffer in trypomastigotes and epimastigotes and by Ba(2+) in epimastigotes and amastigotes. The presence of K(+) channels in amastigotes and a sodium pump in trypomastigotes, in addition to the H(+)-ATPase, could provide important new targets for trypanocidal drug development.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training

Summary Eight male subjects (mean age 24.1±2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the acute phase reactants were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C₁-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.This research was supported by the Bundesinstitut für Sportwissenschaften (Köln-Lövenich)     

Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.Infection by the protozoan Trypanosoma cruzi is a cause of significant morbidity and mortality in Central and South America (15, 34). Mammalian infection is initiated during feeding of the triatomine vector and contamination of the bite wound with infective metacyclic trypomastigotes present within the insect excreta. Once introduced, parasites bind to and invade host cells, where they differentiate and replicate as intracellular amastigotes. Replicating intracellular amastigotes differentiate into trypomastigotes, burst out of their host cells, and invade uninfected host cells. The clinical sequelae of T. cruzi infection include myocarditis, known as Chagas'' heart disease, which is a leading cause of cardiac disease in Latin and South America; megaorgan syndromes; and central nervous system disease in immunosuppressed or pediatric patients.A variety of parasite molecules are known to facilitate the binding of T. cruzi to host cells (4). Soluble and membrane-bound trans-sialidases (8) transfer host sialic acid to parasite surface ligands, including mucin-like molecules (30). Other parasite surface proteins have also been implicated, either positively (29) or negatively (19), in host infection. Invasion of nonphagocytic host cells appears to be an active process involving lysosome recruitment (27), requiring intracellular signaling involving Ca²⁺ mobilization (4, 35). A family of cell surface-localized, zinc-dependent metalloproteases (also known as GP63 proteins, major surface proteases, or leishmanolysins) are expressed by trypanosomes and Leishmania species. In Leishmania, they serve as ligands for host cell attachment and protect the parasite from intraphagolysosomal degradation (6, 20). In the bloodstream form of the African trypanosome Trypanosoma brucei, they function to release variant surface glycoproteins from the cell surface during antigenic variation (17), but they do not function this way in the insect form (12). T. cruzi genes encoding GP63 homologues (TcGP63) are differentially expressed stage specifically, being more abundant in amastigotes than in epimastigotes or trypomastigotes (14). Anti-peptide antibodies against an epitope present in a subset of TcGP63 proteins (termed TcGP63 I) recognized the protein at all life stages and were shown to inhibit trypomastigote infection of host cells (9). Complete genome and proteome analyses of T. cruzi have revealed a vast array of TcGP63 genes (174 “true” copies), encoding at least 29 different proteins. Many of these are expressed at multiple life stages, while some are stage specific (1).To further understand the pattern of expression and functional role of GP63 proteins in T. cruzi, we developed a polyclonal antiserum against recombinant TcGP63 and used this reagent to study the differential expression, subcellular locations, and posttranslational modifications of TcGP63 in both the mammalian and insect forms of the parasite. This antiserum recognizes a different subset of TcGP63 than an anti-peptide antiserum described previously, and comparison of these two antisera demonstrates that different TcGP63 proteins undergo differential stage-specific posttranslational processing. Infection inhibition studies using both antisera show that each independently contributes to host cell infection. Our characterization of GP63 proteins of T. cruzi paves the way for further biochemical and functional analysis of this family of metalloproteases.     

Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi

Antiserum to LPPG, a lipopeptidophosphoglycan originally described on the surface of Trypanosoma cruzi epimastigotes of the Y strain, and antibodies to furanoic galactose (gal_f) were obtained in rabbits. A micromethod for the extraction and purification of LPPG from a limited amount of parasites is described. Analysis by Western blots of the purified glycoconjugate probed with both antisera confirmed the presence of gal_f-containing LPPG-like molecules in 10 different strains and clones of T. cruzi. An analogous approach indicated that trypomastigotes also contain LPPG-like components. Quantitation experiments allowed to calculate an average value of 1.0 × 10⁷ LPPG molecules per epimastigote cell and 0.16 × 10⁷ LPPG-like molecules per trypomastigote cell.

Immunoelectron microscopy has shown a homogeneous distribution of LPPG on the surface of epimastigotes. The trypomastigote population, however, is highly heterogeneous with no more than 15% of the parasites being labeled by the anti-LPPG serum. Intense labeling has also been found in vesicles inside the epimastigote and trypomastigote forms. The distribution of gal_f epitopes among glycoconjugates of epimastigotes and trypomastigotes was further investigated. It was shown that gal_f units in epimastigotes are bound to low molecular mass compounds which co-migrate with LPPG whereas in trypomastigotes they have been found in both low molecular mass LPPG-like molecules and glycoproteins of 80–90 kDa. Direct chemical evidence for the presence of gal_f residues in the N-linked oligosaccharide chains of these surface glycoproteins has been obtained. Finally, the natural antigenicity of LPPG and gal_f in chronic Chagas' disease was investigated. It was found that all chronic chagasic sera investigated recognize this glycoconjugate and that an important part of such recognition can be attributed to gal_f residues. Furthermore, no correlation among reactivity to LPPG, strain zymodeme and clinical forms of the disease was found.     

Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas’ disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V_max = 5.34 ± 0.54 nmol/min per mg of protein; K_m = 0.38 ± 0.01 mM) when compared to intracellular epimastigotes (V_max = 2.18 ± 0.20 nmol/min per mg of protein; K_m = 0.39 ± 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.     

Quantitative receptor autoradiography of eight different transmitter-binding sites in the hippocampus of the common marmoset, Callithrix jacchus

The regional and laminar distributions of eight different transmitter-binding sites were measured in the marmoset hippocampus by means of quantitative in vitro receptor autoradiography. Receptors for 5-HT₁, l-glutamate, N-methyl-d-aspartate (NMDA) and GABA_A were similarly distributed. The highest concentrations of these receptors were found in the pyramidal layer of CA1 and the molecular layer of the dentate gyrus. The 5-HT₂ receptors showed the highest concentrations in the oriens layer of CA2. The highest concentrations of muscarinic M1 receptors were seen in the pyramidal layer of CA1. Muscarinic M2 receptors were most densely concentrated in the pyramidal layers of CA1, CA2 and CA3. The noradrenergic ₁ receptors were most densely packed in the radiatum-lacunosum-molecular layer of CA2 and the molecular layer of the dentate gyrus. Statistically significant co-distributions of serotoninergic, glutamatergic and GABAergic receptors point to possible interactions between these receptor systems in the same hippocampal regions and layers. Comparisons of marmoset distribution patterns for GABA_A, NMDA, l-glutamate and 5-HT₁ receptors with those in human hippocampi and those of other primates showed similarities between them. Clear differences in the patterns of ₁, M1, M2 and 5-HT₂ receptors could be seen between marmoset and human hippocampi, indicating a high degree of species specificity in a presumably conservative brain region. More similarities, however, could be found between marmoset and human hippocampi than between those of rat and human brains, especially in relation to 5-HT₁ and GABA_A receptors and l-glutamate-binding sites. In addition to the functional significance of receptor distribution patterns, such studies represent a valuable tool for the analysis of neurochemical aspects of brain evolution.Abbreviations 5-HT 5-Hydroxytryptamine - GABA gamma-aminobutyric acid - NMDA N-methyl-d-aspartate - NMS N-methyl-scopolamine - CA1-CA4 subfields of the cornu ammonis - DG dentate gyrus - SEM standard error of the mean - AChE acetylcholinesterase - ChAT choline acetyltransferase - GAD-IR glutamate-decarboxylase-immunoreactive     

Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi

Croton cajucara is a plant found in the Amazon region and is known for its medicinal properties. The effects of the methanolic extract of the stem bark of C. cajucara (MCC) and of the isolated terpenes, trans-dehydrocrotonin (t-DCTN) and acetyl aleuritolic acid (AAA), were investigated using four isolates of Trypanosoma cruzi. In assays with trypomastigotes, the extract was more active than the isolated compounds, presenting IC₅₀ in the range of 10 to 50 μg/mL. The trypanocidal effect of MCC, AAA and benznidazole was significantly higher in the GLT291 and C45 strains, which were recently isolated from wild animals. MCC and AAA caused a dose-dependent inhibition of epimastigote proliferation. In assays using intracellular amastigotes, AAA and MCC reduced the percent of infection and the endocytic index after 96 h of treatment, at concentrations that were non-toxic to the host cells. MCC inhibited the trypanothione reductase pathway in both epimastigotes and trypomastigotes of all the subpopulations. The absence of AAA activity on the trypanothione reductase pathway in epimastigotes of Dm28c suggests heterogeneity of the biochemical profile between this clone and the three strains. Epimastigotes and trypomastigotes (GLT291) were treated for 24 h with MCC or AAA, and both induced alterations of the plasma membrane, while AAA-treated epimastigotes also displayed mitochondrial damage.     

Stage specific gene expression precedes morphological changes during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes to metacyclic trypomastigotes of the Trypanosoma cruzi clone Dm 28c has been studied in an in vitro system consisting of artificial triatomine urine supplemented with newborn calf serum. The comparison of morphological data with gene expression products, as judged by the proteins synthesized during differentiation, has shown that stage specific gene activation precedes by far the morphological changes of differentiating cells. Immunoprecipitation of differentiating cell antigens with a trypomastigote stage specific antiserum has shown that although the morphological differentiation process takes six days to be completed, epimastigotes start to express the Mr 86 000 and the 78 000 trypomastigote antigens within the first 12 h of induction.     

Effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) and thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) essential oils on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> (Protozoa: Kinetoplastida) growth and ultrastructure

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)₅₀ was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC₅₀/24 h = 175 μg/ml) and also induced trypomastigote lysis (IC₅₀/24 h = 115 μg/ml). Thyme essential oil presented IC₅₀/24 h values of 77 μg/ml for epimastigotes and 38 μg/ml for trypomastigotes, while treatment with thymol resulted in an IC₅₀/24 h of 62 μg/ml for epimastigotes and 53 μg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.     

The activity of ketoconazole and other azoles against Trypanosoma cruzi: biochemistry and chemotherapeutic action in vitro

Trypanosoma cruzi epimastigotes in culture medium, and amastigotes and trypomastigotes in cultured human diploid lung cells were exposed to the antimycotic agent ketoconazole and their growth and/or sterol biosynthesis observed. Propagation of epimastigotes and amastigotes was impaired by concentrations of ketoconazole achievable in human serum, and amastigotes were more sensitive than were epimastigotes. Epimastigotes and trypomastigotes (non-dividing stage) displayed changes in their membrane sterol content such that the amounts of normal, end-product sterols (ergosterol, ergosta-5,7-dien-3 beta-ol, 24-ethylcholesta-5,7,22-trien-3 beta-ol, 24-ethylcholesta-5,7-dien-3 beta-ol) were notably decreased and the amounts of 14 alpha-methyl sterol precursors of these sterols (24-methylenedihydrolanosterol, obtusifoliol, lanosterol) were increased. Other azole drugs, itraconazole and fluconazole, when tested on epimastigotes, evoked the same qualitative pattern of changes in free sterols. Itraconazole was nearly as potent as ketoconazole, but fluconazole was significantly less potent. The nature of the sterols found in T. cruzi and the actions of azole drugs on their biosynthesis were similar in many respects to those observed in fungi and in Leishmania species. By analogy, it would seem that the primary mechanism of action of azole drugs on T. cruzi life-cycle stages is the impairment of the cytochrome P-450 sterol 14 alpha-demethylase. The consequent loss of normal sterols and accumulation of 14 alpha-methyl sterols may be responsible for the coincident retardation or cessation of growth.