Glycolipid components of epimastigote forms of Trypanosoma cruzi

Glycosphingolipids were isolated from a lipid extract of epimastigote forms of Trypanosoma cruzi via Florisil and silicic acid column chromatography. The carbohydrate components of neutral glycolipid consisted of mannose and galactose in a ratio of 1:2. The fatty acids of the glycolipid were analyzed by gas liquid chromatography-mass spectrometry (g.l.c.-m.s.). Normal and 2-hydroxy fatty acids were found. The sphingosine bases were C18 dihydrosphingosine and 17-methyl sphingosine.     

Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.

Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.     

Electronmicroscopic observations on lysis of Trypanosoma cruzi epimastigote by normal rabbit serum.

The lytic effects of serum from a non-immunized rabbit on epimastigotes of Trypanosome cruzi were studied by electronmicroscopy. The first detectable change was the appearance of a fuzzy deposit over the whole surface of the epimastigote. Soon after this, pellicular microtubules disappeared without change of axonemal microtubules. Circular lesions were observed by negative staining, corresponding to the lesion of antibody-mediated lysis caused by complement.     

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.     

Study of Trypanosoma cruzi epimastigote cell death by NMR-visible mobile lipid analysis

Cell death mechanisms in Trypanosoma cruzi have not been disclosed in detail though different conventional techniques have been used in the classification of parasite-cell death type. Nuclear magnetic resonance (NMR) has successfully been used as a tool to evaluate the onset of apoptosis in a number of higher eukaryote-cell models analysing the ratio of CH(2)/CH(3) integration from the visible mobile lipids (VML). Surprisingly, this versatile non-invasive spectroscopy technique has never been employed with this purpose in T. cruzi. In the present study it is shown that under different parasite death-conditions the ratio CH(2)/CH(3) varied drastically. Thus, T. cruzi epimastigotes in apoptotic conditions increase significantly this ratio while in necrotic as well as in autophagic situations the parasites maintain the VML, CH(2)/CH(3) ratio, in normal values. Additionally, other VML markers commonly used in these studies, such as the change in the region of methyl-choline moiety, -N(+)(CH(3))(3), exhibited different particular patterns according to the type of cell death. Our results suggest that the (1)H NMR-VML technique is an adequate tool to discriminate different T. cruzi death pathways.     

Crossed-immunoelectrophoretic analyses of Trypanosoma cruzi epimastigote, metacyclic, and bloodstream forms

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.     

Characterization of an adenylyl cyclase activity in particulate preparations from epimastigote forms of Trypanosoma cruzi.

Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.     

Trypanosoma cruzi: cell biological behavior of epimastigote and amastigote forms in axenic culture

A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G1 cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.     

Primary structure of the oligosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi

The lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi is composed of a glycan linked through a non-N-acetylated glucosamine residue to an inositol phosphorylceramide. Using conventional analysis techniques, including 1H, 13C, and 31P NMR spectroscopy and negative ion fast atom bombardment mass spectroscopy, the structure of the carbohydrate-containing part of the molecule is determined as: (Sequence: see text). There is uncertainty as to which 2-O-substituted alpha-D-Manp unit is attached the side chain or whether it is distributed between the two units. Some of the structures lack the Galf side chain. The inositol unit is linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion were lignoceric acid and sphinganine.     

Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by "4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca²⁺-dependent while lysis is delayed in the absence of exogenous Ca²⁺. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.Abbreviations TPCK Tos-Phe-CH₂Cl,1-chloro-4 phenyl-3-L-tosylamidobutan-2-one - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - FCS Fetal Calf Serum     

Trypanosoma cruzi: protection of mice with epimastigote antigens from immunologically different parasite strains

Antigens, prepared by the aid of pressure, from epimastigotes of strains of T. cruzi belonging to the different immunological groups described, conferred equal protection in mice against lethal infections of T. cruzi trypomastigotes of the T strain, which belongs to one of those immunological groups. Humoral antibodies were detected by the direct agglutination and immune fluorescent tests in all the immunized groups. The B and T strains produced earlier antibody responses than G and L strains. The weakest antibody response was induced by antigens obtained from the L strain. All the immunized mice sacrificed 21 days after challenge infection showed prominent inflammatory reactions at the tissue level, as well as free amastigote-like bodies. Four months after challenge injection, myocardium, liver, and spleen appeared histologically normal when compared to uninfected control mice. However, histological alterations were detected usually in striated muscle. The latter tissue seemed to be the best to check residual infections.     

Relationship between DNA methylation and cell proliferation in Trypanosoma cruzi.

5-Azacytidine treatment of T. cruzi epimastigotes in culture induces active cell proliferation. This effect was detected as an increase in the cell number and [3H-methyl]thymidine incorporation into DNA. 5-Azacytidine does not alter other metabolic parameters. We have previously demonstrated that 5-azacytidine induces DNA hypomethylation in T. cruzi. Accordingly, we suggest that this chemical modification may be related to the control of T. cruzi cell division.     

Trypanosoma cruzi: activities of lapachol and alpha- and beta-lapachone derivatives against epimastigote and trypomastigote forms

Derivatives of natural quinones with biological activities, such as lapachol, alpha- and beta-lapachones, have been synthesized and their trypanocidal activity evaluated in vitro in Trypanosoma cruzi cells. All tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity as compared with current trypanocidal drugs, nifurtimox and benznidazole. The results presented here show that the anti-T. cruzi activity of the alpha-lapachone derivatives can be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress through redox cycling or production of electrophilic metabolites are the potential biological mechanism of action for these synthetic quinones.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.