Trypanosoma cruzi: Flow Cytometric Analysis of Developmental Stage Differences in DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2–12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4–8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5–13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.     

Trypanosoma cruzi: differences in cell surface interaction of circulating (trypomastigote) and culture (epimastigote) forms with macrophages

Characteristics of the association of circulating (trypomastigote) and cultured (epimastigote) forms of Trypanosoma cruzi with macrophages were studied. Treatment of mouse macrophages with the anti-microfilament drug cytochalasin D severely reduced the ability of these cells to bind either trypomastigotes or epimastigotes. Instead, treatment with the antimicrotubule drug colchicine or 2-deoxyglucose afforded differential effects because epimastigote but not trypomastigote association with the macrophages was significantly inhibited. Prior treatment of epimastigotes with either trypsin or neuraminidase decreased their uptake by macrophages whereas treatment of trypomastigotes with either enzyme increased it. Pretreatment of macrophages with neuraminidase did not affect epimastigote uptake but reduced that of trypomastigotes. Pretreatment of macrophages with trypsin reduced the uptake of both forms of the parasite. However, quantitative differences in the extent of such reduction were noted when varying concentrations of trypsin were used, epimastigote uptake being more drastically affected. These results suggest that the initial interaction of virulent circulating trypomastigote and the attenuated cultured epimastigote forms of T. cruzi to macrophages may involve attachment via different surface structures.     

Characterization of host cell-derived membrane proteins of the vacuole surrounding different intracellular forms of Trypanosoma cruzi in J774 cells. Evidence for phagocyte receptor sorting during the early stages of parasite entry.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, exhibits developmental regulation of virulence. Although both noninfective epimastigote and infective trypomastigote stages of T. cruzi enter phagocytic cells via the formation of a parasitophorous vacuole (PV), only the latter developmental stages survive ingestion and perpetuate the infection. To determine whether the membrane composition of PV surrounding these different stages might contribute to differences in the outcome of infection, we identified selected membrane constituents by immunofluorescence and intracellular radioiodination, and studied their incorporation into PV. Complement receptors (CR3) are incorporated preferentially into the PV membrane surrounding serum-opsonized epimastigotes but not culture-derived metacyclic trypomastigotes. FcR are not preferentially incorporated into PV membranes unless epimastigotes or culture-derived metacyclic trypomastigotes are opsonized with anti-T. cruzi antibody. PV surrounding either parasite stage contain beta 1 integrins and lysosomal membrane glycoproteins (lgp). These results indicate that the plasma membrane glycoproteins incorporated into the surrounding PV membrane differ depending upon the stage of parasite being internalized, and that these differences reflect, at least in part, selective ligation of cell surface receptors mediating uptake. Furthermore, they imply that although virulent trypomastigote stages may avoid host cell uptake by conventional phagocytic receptors, i.e., CR3 or FcR, they do not escape fusion with an lgp-containing vacuole where they could still be exposed to lysosomal antimicrobial mechanisms.     

Trypanosoma cruzi: flow cytometric analysis. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence

Total DNA/organism was determined by flow cytometry on stocks of 33 single-cell-isolate clones and one strain of mithramycin-stained Trypanosoma cruzi. Interstrain differences in mean total DNA/group of 34% and interclone differences in total DNA/organism of 41% were found. Microspectrofluorometric analyses of the trypomastigote stage of selected clones confirmed the flow cytometry data and indicated that the total DNA/organism differences were due to differences in DNA of both the nucleus and kinetoplast with the nucleus being the major contributing factor. These data imply that the potential for genetic diversity in T. cruzi may be very large.     

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.     

Ultrastructural localization of basic proteins in Trypanosoma cruzi.

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react.     

Biological action of pyrazolopyrimidine derivatives against Trypanosoma cruzi. Studies in vitro and in vivo

The capacity of 54 different pyrazolo(3,4-d) or (4,3-d)pyrimidine derivatives to inhibit Trypanosoma cruzi epimastigote and trypomastigote multiplication, and for some of them its chemotherapeutic activity, was evaluated. Six pyrazolo(3,4-d)pyrimidines showed inhibitory activity against epimastigote forms, 4-aminopyrazolo(3,4-d)pyrimidine being the most active, 5-fold more so than 4-hydroxypyrazolo(3,4-d)-pyrimidine. Neither compound was active against freshly isolated trypomastigotes, suggesting biochemical differences between culture and bloodstream forms of T. cruzi. On both epimastigote and trypomastigote forms, 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoA) was about 2-fold more active than 7-hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoB); however, when tested on T. cruzi-infected mice, only FoB exhibited significant chemotherapeutic activity. Previous results suggest that, except for FoB and FoA: (a) pyrazolopyrimidine insensitivity is trypomastigote-specific and (b) drug-insensitivity is lost when trypomastigotes transform into epimastigotes and vice versa.     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.     

Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi

A 1.3 kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.     

Interaction of Trypanosoma cruzi with macrophages: effect of previous incubation of the parasites or the host cells with lectins

The effect of incubation with lectins of the macrophages or two evolutive stages of Trypanosoma cruzi (noninfective epimastigotes and infective trypomastigotes) on the ingestion of the parasites by mouse peritoneal macrophages was studied. Lectins which bind to residues of mannose (Lens culinaris, LCA), N-acetyl-D-glucosamine or N-acetylneuraminic acid (Triticum vulgaris, WGA), beta-D-galactose (Ricinus communis, RCA), N-acetyl-D-galactosamine (Phaseolus vulgaris, PHA; Dolichos biflorus, DBA; and Wistaria floribunda, WFA), fucose (Lotus tetragonolobus, LTA), and N-acetylneuraminic acid (Limulus polyphemus, LPA) were used. By lectin blockage we concluded that, alpha-D-mannose-like, beta-D-galactose and N-acetyl-D-galactosamine (PHA, reagent) residues, located on the macrophage's surface are required for both epi- and trypomastigote uptake, while N-acetylneuraminic acid and fucose residues, impede trypomastigote ingestion but do not interfere with epimastigote interiorization. Macrophages' N-acetyl-D-glucosamine residues are required for epimastigote uptake. On the other hand, from the T. cruzi surface, mannose residues prevent ingestion of epi- and trypomastigotes. Galactose residues participate in endocytosis of trypomastigotes, but hinder epimastigote interiorization. Exposed N-acetyl-D-glucosamine residues are required for uptake of the two evolutive forms. N-acetylneuraminic acid residues on the trypomastigote membrane prevent their endocytosis by macrophages. These results together with those reported previously showing the effect of monosaccharides on the T. cruzi-macrophage interaction, indicate that (a) sugar residues located on the parasite and on macrophage surface play some role in the process of recognition of T. cruzi, (b) different macrophage carbohydrate-containing receptors are involved in the recognition of epimastigotes and trypomastigotes forms of T. cruzi, (c) N-acetylneuraminic acid residues located on the surface of trypomastigotes or macrophages impede the interaction of the parasite with these host cells, and suggest that (d) sugar-binding proteins located on the macrophage surface participate in the recognition of beta-D-galactose and N-acetyl-D-galactosamine residues located on the surface of trypomastigotes and exposed after blockage or splitting off of N-acetylneuraminic acid residues. Some lectins which bind to macrophages and block the ingestion of parasites did not interfere with their adhesion.     

Platelet-activating factor-like activity isolated from Trypanosoma cruzi

Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas' disease, is transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of [14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist, WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating factor-like ligand-receptor system that modulates cell differentiation towards the infectious stage.     

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: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Surface charge of trypanosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility.

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 micrometer-s-1-V-1-cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from staionary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

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.     

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca(2+)-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.     

Growth characteristics in axenic and cell cultures, protein profiles, and zymodeme typing of three Trypanosoma cruzi isolates from Louisiana mammals

Rates of trypomastigote adherence, interiorization, amastigote division in, and trypomastigote release from Vero cells were measured for Trypanosoma cruzi isolates from a dog (Tc-D), opossum (Tc-O), and an armadillo (Tc-A) from Louisiana. Because the Tc-O and Tc-A (wild isolates) trypomastigotes became interiorized rapidly, the media were quickly depleted of trypomastigotes thus reducing the numbers available to adhere to cells. In contrast, the Tc-D trypomastigote interiorization rate was slower. Intracellular amastigote division rate was slower for the Tc-D than the wild isolates. The Tc-D trypomastigotes were released from cells approximately 2 days later than wild isolate trypomastigotes, but twice the number were released. Growth rate for Tc-D epimastigotes in liver infusion tryptose media was faster than that of wild isolates. The doubling times for Tc-D, Tc-O, and Tc-A were 48.0, 69.0, and 67.4 hr, respectively. Soluble parasite extract was produced from epimastigotes of each isolate by freeze/thawing, sonication, and high-speed centrifugation. Proteins were separated on an SDS-PAGE slab gel and stained with Coomassie blue. Although similar bands were present in each preparation, the general pattern of staining was similar only between the Tc-O and Tc-A preparations, which showed some differences from the Tc-D preparation. Each isolate was zymodeme typed using 5 enzymes in lysates produced from epimastigotes of each isolate. Enzymes were separated electrophoretically and stained. Wild isolates showed similar patterns as zymodeme 1 reference stock, whereas the Tc-D isolate produced a pattern that did not resemble any of the reference stocks examined.     

Cytochrome P-450 in culture forms of Trypanosoma cruzi

Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40-60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. Epimastigotes show induced peptides of mol. wt 56,000, 52,000, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.