Growth of Trypanosoma cruzi in Cultures of Chick Embryo Cells, and Effects of Furazolidone and Tris (p-Aminophenyl)Carbonium Chloride

SYNOPSIS. Monolayers of cells of coverslips were produced by culturing known numbers of trypsinized chick cells in growth medium (solution 199 plus 20% calf serum) at 37 C for 2 days. The fluid was then replaced with maintenance medium (solution 199 plus 5% calf serum) containing various known numbers of T. cruzi and the preparations were incubated at 33 C for 5 days; fresh maintenance medium was substituted on the 2nd or 3rd day. The inocula of parasites were obtained from T. cruzi -cell cultures, supplemented with 2% sterile NNN overlay, or from NNN cultures.
The numbers of extracellular parasites, proportions of infected cells, and percentage distribution of infected cells relative to the number of intracellular leishmanial bodies were determined on days 2 or 3 and 5 of parasite cultivation in many experiments. Analyses of the data gave the following results. Extracellular parasites increased 2- to 14-fold during the first 2 or 3 days, depending upon the source and size of the inocula, and 10- to 20-fold during the last 2 or 3 days. Cell infection continued throughout incubation at daily rates of 1.4-4.5%; 8-22% of the cells became infected during the 5 days of incubation. Intracellular growth was reflected most clearly by increases in the proportion of cells having >10 leishmanial bodies. This increase was about 5% daily during the last 2 or 3 days of incubation.
A useful test procedure for assessing the antiparasitic action and chick embryo cell toxicity of drugs is illustrated by data obtained with furazolidone and tris ( p -aminophenyl)carbonium chloride.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by 'in vitro' culture

Abstract By culturing Trypanosoma cruzi epimastigotes in modified Grace's medium with 10% foetal bovine serum, a significant quantity of metacyclic forms could be obtained. Transformation was observed after 8 days of culture, with metacyclic forms reaching 75%. Cultured Vero cells were infected with metacyclic forms and maintained until free-amastigote forms were obtained. Additionally, amastigote-like forms could be obtained by subjecting metacyclic cultures to heat shock. Parasites were grown with glucose as the major carbon source. The metabolites produced and excreted during culture were identified by difference proton nuclear magnetic resonance spectroscopy and quantified by enzymatic methods. The final products of glucose catabolism differed not only quantitatively but also qualitatively for the three major life-cycle stages of T. cruzi . The end products of metabolism produced by epimastigote forms were mainly acetate and pyruvate and, to a lesser extend, l-alanine and ethanol. Differences between epimastigotes and metacyclic forms were only quantitative. However, free amastigotes as well as amastigote-like forms, excreted acetate, glycerol, and pyruvate and to a lesser extent succinate, but no l-alanine or ethanol.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Isolation and purification of amastigotes of Trypanosoma cruzi from cultured vero cells

A method is described for the isolation and purification of the intracellular amastigotes of Trypanosoma cruzi from cultured Vero cells. Host cells were infected with metacyclic forms obtained in Grace's medium. Six days after infection, the cells wer subjected to treatment with trypsin to obtain the intracellular forms. The parasites were collected and purified by Percoll discontinuous gradient centrifugation.     

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.     

Primaquine is lethal for intracellular but not extracellular Trypanosoma cruzi

Primaquine has been used to treat Chagas' disease in humans and has been reported to be active against extracellular Trypanosoma cruzi. Experiments were designed to evaluate the relative activity of primaquine against extra- and intracellular T. cruzi and to determine if primaquine might be combined advantageously with ketoconazole. Primaquine at 0.5 micrograms/ml significantly inhibited T. cruzi replication in infected mouse peritoneal macrophages and also effectively treated infected L929 cells. To examine the effect of primaquine on extracellular organisms, tissue culture T. cruzi were incubated with primaquine for different periods of time and then used to infect macrophages. Incubation with 10 micrograms/ml for 14 hr but not 8 hr significantly inhibited but did not eradicate replication. Incubation of spleen amastigotes or blood trypomastigotes for 2 hr with 10 micrograms/ml did not inhibit replication. Incubation of extracellular tissue culture T. cruzi with primaquine for 2 hr did not potentiate the activity of ketoconazole against intracellular organisms. The combination of primaquine and ketoconazole administered to acutely infected mice significantly decreased parasitemias in comparison to treatment with primaquine or ketoconazole alone. Thus primaquine acts primarily on intracellular rather than extracellular T. cruzi. Primaquine and ketoconazole appear to have additive activity in vivo.     

Biological aspects of the Dm 28c clone of Trypanosoma cruzi after metacyclogenesis in chemically defined media

The biological characterization of the Trypanosoma cruzi clone Dm 28c in terms of its growth in LIT medium, cell-cycle, infectivity to mice and interaction with professional and non-professional phagocytic cells shows that it behaves as a bona fide T. cruzi representant. The biological properties of this myotropic clone do not change according to the origin of the trypomastigote forms (i. e., from triatomines, infected mice, cell-culture or from the chemically defined TAUP and TAU3AAG media). In addition Dm 28c metacyclic trypomastigotes from TAU3AAG medium display a high infectivity level to fibroblasts and muscle cells. Experiments on binding of cationized ferritin to trypomastigotes surface show the existence of cap-like structures of ferritin in regions near the kinetoplast, however the nature and role of these anionic sites remain to be determined. The results indicate that metacyclic trypomastigotes from the Dm 28c clone obtained under chemically defined conditions reproduce the biological behaviour of T. cruzi, rendering this system very suitable for the study of cell-parasite interactions and for the isolation of trypanosome relevant macromolecules.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.     

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.     

Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate

Incubation of Trypanosoma cruzi epimastigotes with Triatoma infestans intestinal homogenate leads to differentiation to the metacyclic trypomastigote. Features of this interaction are presented. The morphogenetic mechanism was triggered almost at once; for the minimum interaction period assayed (15 min), the degree of differentiation achieved in Grace medium by Day 6 was 70.0 +/- 9.0%. Longer interaction periods failed to improve differentiation. The morphogenesis became irreversible at 4 hr after interaction. Epimastigotes incubated for 4 hr with T. infestans intestinal homogenate and then washed reached significant differentiation, while those washed before this time failed to do so. Treatment of epimastigotes with albumin improved the experimental conditions thereby hastening morphogenesis, the same percentage of metacyclics occurring in only 4 days. The factors capable of triggering differentiation were adsorbed by T. cruzi epimastigotes, as expected, but also by Leishmania mexicana and, to a lesser degree, by sheep red blood cells. Once the morphogenetic mechanism had been triggered following interaction of epimastigotes with intestinal homogenate for 15 min, metacyclic forms developed when parasites were transferred to Grace but not to other media. Treatment of epimastigotes with trypsin abolished their capacity to differentiate, which was completely reversed following a 5 hr incubation in LIT medium.     

Inhibition of Trypanosoma cruzi egress from infected fibroblasts is mediated by CD4+ and mu+ immune cells

Trypanosoma cruzi, the causative agent of Chagas disease, is able to reproduce intracellularly in many host cell types while in the mammalian host. Although cellular immunity is known to be important in resistance to infection, the ability of immune cells to interfere with the completion of the intracellular growth cycle of T. cruzi has not been described. Using a tissue culture system to study the parasite growth cycle, we have found that spleen cells from infected mice are able to decrease the number of parasites released from infected fibroblasts. Spleen cells from mice infected for as few as 14 days and as long as 300 days display this inhibitory ability. Parasite egress from infected cells is inhibited by factor(s) released by immune cells during coculture with infected fibroblasts. Immune cell depletion studies indicate that the inhibitory activity requires the presence of both CD4+ T cells and mu+ B cells. These results suggest a direct ability of immune cells to somehow interfere with the completion of the intracellular cycle, and this ability may play a role in control of this parasite.     

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choach -2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.     

Cross-reactivity of vector-borne metacyclic forms of Trypanosoma cruzi with mammalian and culture stages

Metacyclic forms of Trypanosoma cruzi isolated from the hindgut of infected insect vectors (Rhodnius prolixus) were found to be immunologically cross-reactive with cultured epimastigote, amastigote, and metacyclic stages of the parasite as well as with bloodstream trypomastigote forms by direct agglutination and indirect immunofluorescence techniques. Sera specific for each of these forms of the parasite systematically yielded maximal antibody titers when measured against the homologous antigen, indicating that antigenic determinants are shared by all of the developmental forms used in this work. Supporting this conclusion were the significant reductions in anti-insect-derived metacyclic antibody titer caused by absorption with any of the other life stages of T. cruzi. These results are relevant to the potential use of laboratory-grown forms of T. cruzi in vaccination against a natural infection with this parasite.     

Effect of Gossypol on Trypomastigotes and Amastigotes of Trypanosoma cruzi

Bloodstream Trypanosoma cruzi trypomastigotes isolated from infected mice undergo reduction of motility and structural damages after 5 to 45 min exposure to gossypol at concentrations ranging from 5 to 50 μM. When 1% serum albumin is added to the incubation medium, no alterations of parasites are observed, even with 100 μM gossypol. Intracellular T. cruzi amastigotes in infected Vero cell cultures exposed to 5 μM gossypol for 2 h do not show changes. Incubation with 5 μM gossypol for 48 h produces complete disruption of host cells; however, the amastigotes they contain show only mineor alterations. The observations indicate that, in protein-rich media, gossypol is complexed into associations which have no activity on the different forms of the T. cruzi biological cycle.     

Effect of gossypol on trypomastigotes and amastigotes of Trypanosoma cruzi

Bloodstream Trypanosoma cruzi trypomastigotes isolated from infected mice undergo reduction of motility and structural damages after 5 to 45 min exposure to gossypol at concentrations ranging from 5 to 50 microM. When 1% serum albumin is added to the incubation medium, no alterations of parasites are observed, even with 100 microM gossypol. Intracellular T. cruzi amastigotes in infected Vero cell cultures exposed to 5 microM gossypol for 2 h do not show changes. Incubation with 5 microM gossypol for 48 h produces complete disruption of host cells; however, the amastigotes they contain show only minor alterations. The observations indicate that, in protein-rich media, gossypol is complexed into associations which have no activity on the different forms of the T. cruzi biological cycle.     

Purification of metacyclic forms of Trypanosoma cruzi by Percoll discontinuous gradient centrifugation

A method for the purification of metacyclic forms of Trypanosoma cruzi has been developed. Metacyclic forms obtained in modified Grace medium were separated from the epimastigote forms by Percoll discontinuous density gradient centrifugation. Four different osmotic pressures were applied: 160 +/- 10, 260 +/- 10, 310 +/- 10 and 510 +/- 10 mosmol/kg H2O. At 160 mosmol/kg H2O, 100% of the metacyclic forms with a 21.3% yield were found in the interphase 1.120/1.125 g/ml, while 92.7% of the metacyclic forms with a 73.7% yield were found in the interphase 1.115/1.120 g/ml. At 310 mosmol/kg H2O, 100% of the metacyclic forms in the interphase 1.135/1.140 g/ml with a 36.8% yield were obtained. Metacyclic forms purified in this way do not show alterations in their capacity to infect cultures of HeLa cells.     

Sialoglycoproteins and sialoglycolipids contribute to the negative surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi have a net negative surface charge, as determined by direct measurement of the mean cellular electrophoretic mobility. Treatment of the parasites with neuraminidase reduces by 17 and 52% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms, respectively. Neuraminidase-treated cells recover their normal electrophoretic mobility if incubated for 2 h in the presence of fresh culture medium. The recovering process of epimastigotes is almost totally blocked by addition of inhibitors of either protein synthesis (puromycin) or N-glycosidically linked glycoprotein synthesis (tunicamycin). The recovering process of trypomastigotes is not totally inhibited by either puromycin or tunicamycin. Treatment of T. cruzi with trypsin reduces by 11 and 40% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms. Trypsin-treated cells recover their normal electrophoretic mobility if incubated for 4 h in fresh culture medium. The recovering process of trypomastigotes is partially inhibited by puromycin. The results obtained indicate that sialoglycoproteins and sialoglycolipids exist on the surface of T. cruzi, the latter being predominant on the surface of trypomastigotes.     

In vitro effects of citral on trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.