Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

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.     

Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi

The origin of Trypanosoma cruzi slender and broad forms found in the circulation of the mammalian host has remained obscure and, unlike what has been proposed for African trypanosomes, no precise form-function relationship has been ascribed to them. We show here that parasites circulating in the blood of infected animals display a high degree of polymorphism. Around 10% of the forms found circulating in mice during the acute phase of infection were amastigotes, and the other 90% included slender and broad trypomastigotes and intermediate forms between amastigotes and trypomastigotes. Slender trypomastigotes, from blood or cell culture, undergo extracellularly morphological rearrangements in which the parasites become gradually broader and transform into amastigotes. By scanning electron microscopy a progressive internalization of the flagellum and reorganization of the cell shape in a helical fashion were observed in parasites undergoing transformation. After 48 hr of extracellular incubation the parasite population consisted exclusively of amastigotes with a short protruding flagellum. The morphological changes were associated with the expression of different surface antigens defined by monoclonal antibodies: the trypomastigote-specific antigens Ssp-1 (a 100-120-150-Mr glycoprotein), Ssp-2 (a 70-Mr glycoprotein), Ssp-3 (undefined), and Ssp-4, an amastigote-specific surface antigen. Ssp-4 was also detected on intracellular amastigotes (in vitro and in vivo). We conclude that trypomastigotes are programmed to develop into amastigotes whether or not they enter cells, and that the differentiation can occur in the blood of the vertebrate host. These findings raise some questions regarding conventional views on the life cycle of T. cruzi.     

Cell-substrate adhesion during Trypanosoma cruzi differentiation

The transformation of Trypanosoma cruzi epimastigotes to the mammal infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions. Under these conditions, differentiating epimastigotes adhere to a surface before their transformation into metacyclic trypomastigotes. Scanning and transmission electron microscopy of adhered and non-adhered parasites during the metacyclogenesis process show that only epimastigotes and few transition forms are found in the first population, whereas metacyclic trypomastigotes are exclusively found in the cell culture supernatant. PAGE analysis of the [35S]methionine metabolic labeling products of adhered and non-adhered parasites shows that although most of the polypeptides are conserved, adhered parasites express specifically four polypeptides in the range of 45-50 kD with an isoelectric point of 4.8. These proteins might be involved in the adhesion process and are recognized by an antiserum against total adhered parasite proteins. This antiserum also recognized a group of 45-50 kD in the iodine-radiolabeled surface proteins of differentiating cells, providing direct evidence that these components are indeed surface antigens. The results suggest that epimastigotes must adhere to a substrate before their transformation to metacyclic trypomastigotes, being released to the medium as the metacyclogenesis process is accomplished. This could correspond to the process naturally occurring within the triatomine invertebrate host.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Expression of trypomastigote trans-sialidase in metacyclic forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole

Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans-sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic metacyclic trypomastigotes, which correspond to the infective forms derived from the insect vector. Both forms have the same capacity to invade mammalian cells, but cell derived trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of metacyclic forms. Transfected and non-transfected metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected metacyclics, or metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, trypomastigotes and metacyclic forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.     

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.     

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi

The fatty acid composition during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes (metacyclogenesis) was analysed by gas-liquid chromatography and mass spectrometry. Significant qualitative and quantitative changes in the fatty acid composition occurred during incubation of epimastigotes derived from LIT medium in the triatomine artificial urine (TAU). Metacyclogenesis was also followed by alterations in the fatty acid pattern but these were considerably less pronounced when compared to the pattern obtained for TAU-incubated epimastigotes. These results suggest that changes in the lipid composition precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Cyclic AMP and adenylate cyclase activators stimulate Trypanosoma cruzi differentiation

A chemically defined in vitro differentiating condition was used to study the potential role of cyclic AMP (cAMP) and adenylate cyclase activators on the transformation of Trypanosoma cruzi epimastigotes to the infective metacyclic trypomastigotes (metacyclogenesis). It was observed that both addition of cAMP analogs or adenylate cyclase activators to the differentiating medium stimulated the transformation of epimastigotes to metacyclic trypomastigotes. These results were further corroborated by showing that inhibitors of cAMP phosphodiesterase were stimulatory while activators of this enzyme inhibited the metacyclogenesis process. On the other hand, inhibitors of calmodulin inhibited the transformation of epimastigotes to metacyclic trypomastigotes, suggesting that T. cruzi adenylate cyclase might be activated by calmodulin. In addition, the results strongly suggest that guanine nucleotide binding proteins are involved in T. cruzi adenylate cyclase activation. This system may be useful for studying cell differentiation mechanisms in eukaryotes.     

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH(4)Cl, which releases Ca(2+) from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.     

Characterization of a 21 kDa protein from Trypanosoma cruzi associated with mammalian cell invasion

Trypanosoma cruzi genomic database was screened for hypothetical proteins that showed high probability of being secreted or membrane anchored and thus, likely involved in host-cell invasion. A sequence that codes for a 21 kDa protein that showed high probability of being secreted was selected. After cloning this protein sequence, the results showed that it was a ubiquitous protein and secreted by extracellular amastigotes. The recombinant form (P21-His₆) adhered to HeLa cells in a dose-dependent manner. Pretreatment of host cells with P21-His₆ inhibited cell invasion by extracellular amastigotes from G and CL strains. On the other hand, when the protein was added to host cells at the same time as amastigotes, an increase in cell invasion was observed. Host-cell pretreatment with P21-His₆ augmented invasion by metacyclic trypomastigotes. Moreover, polyclonal antibody anti-P21 inhibited invasion only by extracellular amastigotes and metacyclic trypomastigotes from G strain. These results suggested that P21 might be involved in T. cruzi cell invasion. We hypothesize that P21 could be secreted in the juxtaposition parasite-host cell and triggers signaling events yet unknown that lead to parasite internalization.     

Some factors inducing formation of metacyclic stages of Trypanosoma cruzi

Epimastigotes of Trypanosoma cruzi, Peru strain, incubated in Contreras' artificial triatomine urine transformed into metacyclic trypomastigotes within 48 h at 28 degrees C when 10 mM L-proline or L-glutamate was added to the medium. Smaller numbers of metacyclic stages were induced in the presence of glucose or L-alanine. The L-leucine and L-isoleucine, 2 amino acids known to inhibit proline catabolism, inhibited proline-induced metacyclogenesis. Cells gassed with 5% CO2 showed significantly faster rates and higher levels of transformation than those not gassed, therefore indicating the additional importance of CO2 for transformation.     

Immunological characterization of antigens released by Trypanosoma cruzi-infected cells.

Chagas' disease, caused by Trypanosorna cruzi, is characterized by the appearance of pathological lesions in the heart and other tissues during the chronic phase. The mechanisms responsible for such damage are still unclear. In the vertebrate host, T. cruzi replicates intracellularly before transforming from amastigotes into trypomastigotes. The infected host cell then lyses, releasing the cytoplasmic contents and the parasites that shed membrane glycoproteins soon after release. The sum of all these components we have termed released antigen (Rag). We characterized antigens, released in vitro by fibroblasts infected with T. cruzi, obtained by concentrating conditioned serum-free culture media. The results demonstrate that Rag contains a complex protein mixture including stage-specific T. cruzi antigens (Ssp-1, -2, -4), glucose-regulated protein (Grp) 78h, and peptides recognized by the monoclonal antibody 2B10. These peptides exhibit neuraminidase activity and are expressed by intracellular and 10-20% of released trypomastigotes. Additionally, Rag is recognized by sera from T. cruzi-infected mice and human chagasic patients. Rag also stimulates in vitro production of interferon-gamma by splenocytes from resistant C57B1/6 and susceptible BALB/c infected mice and interleukin-4 by splenocytes from BALB/c infected mice. Altogether these results indicate that Rag is immunologically relevant and could contribute to pathogenesis of T. cruzi infection.     

Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.     

Trypanosoma cruzi: characterization of an intracellular epimastigote-like form.

Almeida-de-Faria, M., Freymüller, E., Colli, W., and Alves, M. J. M. 1999. Trypanosoma cruzi: Characterization of an intracellular epimastigote-like form. Experimental Parasitology 92, 263-274. A detailed study of transient epimastigote-like forms as intermediates in the differentiation of Trypanosoma cruzi amastigotes to trypomastigotes inside the host cell cytoplasm was undertaken using the CL-14 clone grown in cells maintained at 33 degrees C. Several parameters related to these forms have been compared with epimastigotes and other stages of the parasite. Consequently, the designation of intracellular epimastigotes is proposed for these forms. Despite being five times shorter (5.4 +/- 0.7 micrometer) than the extracellular epimastigote (25.2 +/- 2.1 micrometer), the overall morphology of the intracellular epimastigote is very similar to a bona fide epimastigote, when cell shape, position, and general aspect of organelles are compared by transmission electron microscopy. Epimastigotes from both sources are lysed by human complement and bind to DEAE-cellulose, in contrast to amastigotes and trypomastigote forms. A monoclonal antibody (3C5) reacts with both epimastigotes either isolated from axenic media or intracellular and very faintly with amastigotes, but not with trypomastigotes. Some differences of a quantitative nature are apparent between the two epimastigote forms when reactivities with lectins or stage-specific antibodies are compared, revealing the transient nature of the intracellular epimastigote. The epitope recognized by 3C5 monoclonal antibody reacts slightly more intensely with extracellular than with intracellular epimastigotes, as detected by immunoelectron microscopy. Also a very faint reaction of the intracellular epimastigotes was observed with monoclonal antibody 2C2, an antibody which recognizes a glycoprotein specific for the amastigote stage. Biological parameters as growth curves in axenic media and inhability to invade nonphagocytic tissue-cultured cells are similar in the epimastigotes from both origins. It is proposed that the epimastigote-like forms are an obligatory transitional stage in the transformation of amastigotes to trypomastigotes with a variable time of permanency in the host cell cytoplasm depending on environmental conditions.