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.     

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: amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells

It is generally accepted that Trypanosoma cruzi trypomastigotes represent the infective forms of the etiological agent of Chagas' disease. However, the invasive capacity of amastigotes and their ability to sustain a complete infective cycle in mammalian cultured cells and hosts has been recently demonstrated. In order to compare the process of cell invasion by these different infective forms, I examined the interactions of trypomastigotes and amastigotes with HeLa cells using a new and simple method that improves parasite-cell interactions and significantly reduces incubation periods. T. cruzi forms were centrifuged onto HeLa cells grown on coverslips and parasite-cell interactions were examined by fluorescence and scanning electron microscopy. As expected, it was observed that all parasite forms attach and eventually enter the cells. However, whereas trypomastigotes preferentially invade HeLa cells at the edges, as has recently been demonstrated for other cell types, the initial steps of amastigote-HeLa cell interaction involve binding and entangling of the parasite to surface microvilli. Thus, different T. cruzi infective forms interact with different cell surface structures that could express different receptors at the HeLa cell membrane.     

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.     

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.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis

Genetic characterisation of Trypanosoma cruzi variants is of foremost importance, due to considerable genetic and biological heterogeneity in the parasite populations. Two major phylogenetic lineages, each highly heterogeneous, have been previously described within this species. Here we characterised a geographically and ecologically diverse sample of stocks representative of the breadth of the known clonal diversity of each major lineage, using random amplified polymorphic DNA with 20 primers and multilocus enzyme electrophoresis at 22 loci. Molecular hybridisation experiments were performed to control the homology of randomly amplified DNA markers. Both sets of data were highly consistent and supported the existence of two major lineages. Additionally, we found that lineage 2 appeared further partitioned into five sharply delineated phylogenetic clusters, each comprising one of the following reference strains: CanIII cl1 (Z3 reference), M5631 cl5, Esmeraldo cl3 (Z2 reference), CL Brener, and MN cl2. The two first clusters were found mainly in sylvatic environments, whereas the three latter were restricted to domestic transmission cycles and were only collected South to the Amazon Basin. In contrast, lineage 1, which included Miles' Z1 reference strain X10 cl1, was not further subdivided and was encountered across the entire endemic area, in both domestic and sylvatic cycles. Thus, T. cruzi appeared to be subdivided into six discrete typing units, or DTUs, exhibiting distinct geographic and ecological ranges. Reliable diagnostic markers for the two major lineages and the five smaller DTUs of lineage 2 are described, and correspondence with previous classifications of T. cruzi genotypes is given in order to help communication on T. cruzi phylogenetic diversity.     

Trypanosoma cruzi: antibody-dependent killing of bloodstream trypomastigotes by mouse bone marrow-derived mast cells and by mastocytoma cells

Two different populations of mast cells, that is, mastocytoma cells (P815) that were maintained either in vitro or in vivo, and mast cells obtained by differentiation of bone marrow precursor cells (MMC) in conditioned medium, were used as effector cells in antibody-dependent cytotoxic reactions (ADCC) against bloodstream trypomastigotes (BT) of Trypanosoma cruzi. The assay consisted of incubating effector cells with parasites that had been previously sensitized with immune mouse sera, immune IgG isotypes, or with medium. After the incubation period, the number of live BT was assessed. It was found that (a) cytotoxicity is antibody dependent; (b) the main isotypes involved are IgG1, IgG2a, and IgG2b; (c) both types of mast cells (mastocytoma and MMC cells) are equally efficient in killing BT; (d) mastocytoma cells degranulated by pretreatment with compound 48/80 are still able to effect ADCC; (e) on optical microscope examination, large numbers of parasites were often seen attached to the cells, but only when anti-T. cruzi antibodies were present; and (f) on electron microscope examination, no integral or ruptured parasites were seen inside the cells. We conclude that both T dependent and T independent mast cells are capable of mediating ADCC by a mechanism that is probably not dependent on granule extrusion.     

Evolution of Trypanosoma cruzi: clarifying hybridisations,mitochondrial introgressions and phylogenetic relationships between major lineages

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIV_S and TcIV_N). In addition, we collected evidence that TcIII received TcIV_S kDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIV_S in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIV_S kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.     

Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate

Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation     

Trypanosoma cruzi: Serum levels of nitric oxide and expression of inducible nitric oxide synthase in myocardium and spleen of dogs in the acute stage of infection with metacyclic or blood trypomastigotes

The participation of nitric oxide (NO) in the control of blood parasitemia and parasitism during the acute phase of infection in dogs inoculated with blood trypomastigotes (BT) or metacyclic trypomastigotes (MT group) of Berenice-78 Trypanosoma cruzi strain has been evaluated. Animals of the MT group (n = 4) presented increased levels of serum NO throughout the infection when compared with the BT (n = 4) or control (n = 4) groups, and a delay in parasitemia peak compared with the BT group. In spleen fragments, tissue parasitism was not observed but the MT group presented larger areas associated with inducible NO synthase (iNOS) in relation to BT and control groups. Heart fragments of MT-infected animals exhibited comparatively low tissue parasitism and high iNOS expression, while animals of the BT group presented high inflammatory infiltrate, high tissue parasitism and low iNOS expression. These results indicate that the source of inoculum can interfere with the development of the acute phase of Chagas disease, and may also trigger a distinct parasite-host interaction during this phase.     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

Protease expression analysis in recently field-isolated strains of Trypanosoma cruzi: a heterogeneous profile of cysteine protease activities between TC I and TC II major phylogenetic groups

Protease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50-60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.     

Challenge of Trypanosoma cruzi chronically infected mice with trypomastigotes activates the immune system and reduces subpatent parasitemia levels

Challenge of 1-yr Trypanosoma cruzi chronically infected mice with trypomastigotes results in a consistent reduction of parasite dissemination that correlates with spleen activation and increase in the anti-T. cruzi effector immune mechanisms. That is, parasite challenge results not only in elimination of the inoculum but also in a drastic decrease in basal subpatent parasitemia levels as revealed by transferring blood samples to immunosuppressed mice. Parasite elimination correlated with (1) a brief and intense burst in the ability of spleen cells to produce interferon-gamma, (2) an increase in total IgG2a-producing spleen cells, (3) higher parasite-specific IgG2a serum levels, and (4) an accumulation of non-B, non-T class II+ cells in the spleen. Furthermore, challenged, chronically infected mice had increased numbers of B, CD4+, and CD8+ large spleen cells. Besides reinforcing the activation of protective Th1 effector mechanisms, challenge with T. cruzi also induced Th2 effector molecules, such as interleukin (IL)-10 and IL-4, and IL-4-dependent IgG1. Our results are the first evidence that the immune system of T. cruzi chronically infected mice can be optimized in its ability to restrict parasite dissemination, opening the possibility that therapeutic vaccination could be used to reduce the parasite load and pathology of patients with chronic Chagas' disease.