Comparative studies on surface antigenicity of Trypanosoma cruzi trypomastigotes from infected mouse blood and infected L-cell cultures

Differences in the antigenicities of surface components of blood-form trypomastigotes and trypomastigotes derived from L-cell cultures were studied by agglutination and indirect immunofluorescent tests on living parasites using various antisera from rabbits and mice. Antisera from rabbits immunized with L-cell-derived trypomastigotes and antisera obtained from rabbits infected with L-cell-derived trypomastigotes showed similar titers in both the agglutination and immunofluorescent test. Moreover, both antisera exhibited higher titers against trypomastigotes derived from L-cell cultures than against blood-form trypomastigotes. No detectable agglutination titer against either blood-form or L-cell-derived trypomastigotes was observed with sera from (a) mice infected with blood-form trypomastigotes after previous immunization with blood-form trypomastigotes, (b) mice infected with blood-form trypomastigotes and then treated with Lampit, or (c) mice infected with slightly less virulent trypomastigotes from L-cells. However, detectable and almost equal titers were observed with sera from (a), (b) and (c) in indirect immunofluorescent tests. Mouse sera also exhibited higher titers against trypomastigotes derived from L-cells than against the blood-form type. However, mouse sera showed more pronounced differences than rabbit sera. These results suggest that there may be two types of trypomastigotes in infected animals and that the surface components of blood-form trypomastigotes have lower antigenicity.     

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.     

Attachment of Trypanosoma cruzi to host cells: a monoclonal antibody recognizes a trypomastigote stage-specific epitope on the gp 83 required for parasite attachment.

A set of monoclonal antibodies against the purified surface gp 83 of T. cruzi trypomastigotes was produced and the ability of these monoclonals to inhibit the attachment of trypomastigotes to heart myoblasts was investigated. Western blots of solubilized trypomastigotes, epimastigotes or amastigotes probed with this set of monoclonal antibodies show that the gp 83 is present in invasive trypomastigotes, but not in non-invasive epimastigotes or amastigotes. One monoclonal antibody (Mab 4A4) from this set inhibits the attachment of trypomastigotes to heart myoblasts, whereas the others (MAbs 2H6, 4B9, 2D11) do not. These results show that the Mab 4A4 recognizes an epitope on the gp 83 of invasive trypomastigotes required for parasite binding to host cells.     

Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes

A rabbit antibody to the neuraminidase of the infective form of Trypanosoma cruzi identifies a subpopulation of trypomastigotes that expresses neuraminidase. Complement-mediated lysis by the antibody selectively destroys 30 to 40% of the trypomastigotes, supporting the conclusion that the immune antibody binds to a subset of parasites. The trypomastigotes that react with the immune antibody are the only ones expressing neuraminidase because the trypomastigotes that survive complement-mediated lysis are depleted of neuraminidase activity. The enzyme seems to negatively modulate infection in vitro, since infection of host cells by trypomastigotes is enhanced when neuraminidase activity is blocked by antineuraminidase antibody; infection is also enhanced when the infecting trypomastigotes have been depleted of parasites that express neuraminidase. Addition of exogenous neuraminidase (from Vibrio cholerae) to trypomastigotes treated with immune antibody, reverts the enhancement observed when infection takes place in the presence of antibody to T. cruzi neuraminidase only. Addition of V. cholerae neuraminidase in the absence of immune antibodies has no effect on infection. These results show that T. cruzi neuraminidase depresses infection and also suggest that sialic acid is involved in the parasite-host cell interaction. The antibody to T. cruzi neuraminidase recognizes on the surface of live trypomastigotes a set of proteins with high m.w. (165,000 to 200,000) and also two antigens of 79,000 to 82,000. The high m.w. proteins appear to be associated with neuraminidase activity as shown by renaturation experiments of released enzyme fractionated on a sodium dodecyl sulfate-polyacrylamide gel.     

Incubation in Mice Provides a Signal for the Differentiation of Trypanosoma cruzi Epimastigotes to Trypomastigotes1

The differentiation of Trypanosoma cruzi epimastigotes into trypomastigotes was studied in diffusion chambers sub-cutaneously implanted in mice. Using epimastigotes of the Tulahuén strain, transformation was first evident at 16 h after implantation and reached its maximum (92% trypomastigotes) by 24 h. Shortly before their differentiation into trypomastigotes, epimastigotes were found to develop resistance to lysis by the alternative pathway of complement. Furthermore, implantation of stationary-phase (as opposed to log-phase) parasites resulted in the accumulation of large numbers of complement-resistant epimastigotes in the chambers. These observations suggest that epimastigotes pass through a complement-resistant transitional stage before differentiating into trypomastigotes and that transformation may require cell division. In a further series of experiments, epimastigotes recovered 7 h after implantation in mice were found to differentiate into trypomastigotes when cultured in vitro for an additional 17 h at 37°C. This observation indicates that the events which trigger the morphologic transformation of epimastigotes into trypomastigotes can be dissociated operationally from the differentiation process itself.     

The Attraction of Trypanosoma cruzi to Vertebrate Cells In Vitro

SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruzi trypomastigotes to vertebrate cells in vitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T. cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T. cruzi trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of pO₂ or pCO₂ gradients in the milieu around the cells.     

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.     

Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.     

Binding of complement to trypomastigotes of a Brazil strain of Trypanosoma cruzi: evidence for heterogeneity within the strain.

Binding of the complement components C3 and C5 to epimastigote and trypomastigote stages of the Brazil strain of Trypanosoma cruzi was examined using radioligand binding and flow cytometric assays. Fibroblast-derived trypomastigotes bound approximately 40% fewer molecules of [125I]C3 per parasite than did epimastigotes. The predominant molecular species of C3 deposited on fibroblast-derived trypomastigotes was the inactive form iC3b. Addition of parasite-specific antisera failed to enhance the number of molecules of [125I]C3 per parasite or the proportion of active to inactive C3b. Flow cytometric studies revealed that only 50% of trypomastigotes (fibroblast-derived or blood-form) bound C3. In contrast to results of the [125I]C3 binding studies, flow cytometric analysis showed that the percentage of trypomastigotes binding C3 actually increased upon incubation with parasite-specific antisera. C5 was found also to bind to only a percentage of trypomastigotes.     

Trypanosoma cruzi: cell type dependent distribution of a tubulin domain in mammalian stages

The distribution of tubulin domains in the mammalian stages of Trypanosoma cruzi was investigated by using monoclonal antibodies elicited against bovine brain tubulin. Western blotting performed on T. brucei trypomastigotes and T. cruzi epimastigotes showed that the monoclonal antibodies 16D3 and 24E3 reacted only with tubulin in these cell types. Indirect immunofluorescence revealed that, whereas 16D3 stained all microtubules, including subpellicular microtubules, the epitope defined by 24E3 was found in only a part of the tubulin pool of amastigotes and intermediate stages infecting murine fibroblasts and of broad trypomastigotes; the staining was limited to the basal bodies and the distal region of the flagellar adhesion zone in these developmental forms. By contrast, slender trypomastigotes did not exhibit any reaction with 24E3. These results are consistent with a transformation of broad trypomastigotes into slender trypomastigotes during which the tubulin domain recognized by 24E3 would undergo modifications leading to its complete masking in slender forms. The morphogenesis of the mammalian stages of T. cruzi would involve modifications of the tubulin molecule.     

Host-cell attachment by Trypanosoma cruzi: identification of an adhesion molecule

We have identified an 83 kDa surface glycoprotein in T. cruzi trypomastigotes which specifically binds to rat heart myoblasts. The binding of this molecule to myoblasts is inhibited by excess unlabeled material and saturable. Antibodies against the cell surface of insect trypomastigotes, blood trypomastigotes and produced during human infection recognize the 83 kDa glycoprotein adhesion molecule by immunoblotting, indicating that this molecule that mediates this critical step is immunogenic and is a candidate for vaccination against Chagas' disease.     

Identification and Partial Purification of a Stage-Specific 33 kDa Mitochondrial Protein as the Alternative Oxidase of the Trypanosoma brucei brucei Bloodstream Trypomastigotes

ABSTRACT. The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.     

Polymorphism in trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets.

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense, although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.     

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.     

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.     

Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.     

The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi

The high resistance of Trypanosoma cruzi trypomastigotes, the causal agent of Chagas' disease, to complement involves several parasite strategies. In these in vitro studies, we show that T. cruzi calreticulin (TcCRT) and two subfragments thereof (TcCRT S and TcCRT R domains) bind specifically to recognition subcomponents of the classical and lectin activation pathways (i.e., to collagenous tails of C1q and to mannan-binding lectin) of the human complement system. As a consequence of this binding, specific functional inhibition of the classical pathway and impaired mannan-binding lectin to mannose were observed. By flow cytometry, TcCRT was detected on the surface of viable trypomastigotes and, by confocal microscopy, colocalization of human C1q with surface TcCRT of infective trypomastigotes was visualized. Taken together, these findings imply that TcCRT may be a critical factor contributing to the ability of trypomastigotes to interfere at the earliest stages of complement activation.     

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.     

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote

Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [³H]palmitic acid or [³H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using ³H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A₁, phospholipase A₂, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.     

Relevance of hemin for in vitro differentiation of Trypanosoma cruzi

Simple culture conditions that allow good growth and high yields of trypomastigotes are described. The proportion of metacyclic trypomastigotes increases with the concentration of hemin in the culture medium, reaching a peak of 80% after 10 days with 20 mg hemin/liter.