Trypanosoma cruzi and American Leishmania spp: immunocytochemical localization of a laminin-like protein in the plasma membrane

Patients with Chagas' disease or different clinical forms of American cutaneous leishmaniasis have high antilaminin antibody levels. An immunogold technique employing a specific antilaminin antibody was used in the present study to determine the presence, and define the ultrastructural localization, of laminin-like molecule(s) in American Leishmania spp. and Trypanosoma cruzi. Laminin was found located specifically in T. cruzi trypomastigotes on the external surface of the plasma membrane, close to the sites where the flagellar veil attaches to the plasma membrane. Laminin immunoreactivity was rapidly lost when trypomastigotes were cultured in liquid medium and no reactivity was found in fresh epimastigotes. Promastigotes and amastigotes of American Leishmania spp. also showed a specific localization of laminin immunoreactivity, this being limited to the lips of the flagellar pocket and to the parasitic side exactly opposite to the flagellar exit. These results confirm the presence of a laminin-like molecule(s) in both trypanosomatids, the specific localization suggesting a presently unknown function for this protein.     

The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex.

Okuda, K., Esteva, M., Segura, E. L., and Bijovsky, A. T. 1999. The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex. Experimental Parasitology 92, 223-231. Proliferative forms of Trypanosoma cruzi, amastigotes and epimastigotes, have a cytostome, a specialized structure formed by an invagination of the flagellar pocket's membrane surrounded by microtubules and frequently followed by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm overpassing the nucleus. This structure, together with the flagellar pocket, appears to play an important role in the nutrition of the parasite. We demonstrated that the monoclonal antibody 2C4, made-up against isolated flagellar complex of T. cruzi epimastigotes, recognizes a protein doublet of 76 and 87 kDa in total epimastigotes homogenate. The 76-kDa polypeptide is enriched in the detergent-soluble fraction whereas the 87-kDa polypeptide is highly represented in the insoluble fractions and the purified flagella. Immuno-fluorescence assays show the antigen as a small spot at the flagellar pocket region. Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of flagellar pocket, concentrated in the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of three to four microtubules that appears attached to flagellum, running in the direction of the nucleus. Ultrastructural observations have shown that the posterior region of isolated flagella, corresponding to the level of the flagellar pocket, possesses a microtubular structure compatible with that from the cytostome. The relationship between the cytostome, an endocytic organelle, and the flagellum is here described for the first time.     

Immunogenic Gal alpha 1----3Gal carbohydrate epitopes are present on pathogenic American Trypanosoma and Leishmania

Anti-Gal is a natural antibody present in unusually high concentrations in human sera. It constitutes as much as 1% of circulating IgG and displays a distinct specificity for the Gal alpha 1----3Gal carbohydrate epitope. In the present study, we have found in the sera of patients with Chagas' disease and Leishmania infection anti-Gal titers 10- and 16-fold higher than that of healthy or bacteria-infected individuals. This increase in anti-Gal titer seemed to be the result of a specific immune response toward parasitic Gal alpha 1----3Gal epitopes. Binding studies of affinity chromatography-purified anti-Gal antibodies to Trypanosoma cruzi and American Leishmania parasites indeed demonstrated the presence of Gal alpha 1----3Gal epitopes on these parasites. This finding was supported by the observed binding to the parasites of two additional Gal alpha 1----3Gal recognizing molecules: the mAb Gal-13, and the lectin, Bandeiraea simplicifolia I B4. Furthermore, the binding of both anti-Gal antibody and of the B. simplicifolia I B4 lectin could be inhibited by galactose, and not glucose. In addition, removal of the terminal alpha-galactosyl residues from the parasites by pretreatment with alpha-galactosidase, or the oxidation of the binding epitopes by periodate prevented the subsequent binding of both the antibody and the lectin. A crude leishmanial lipid extract readily bound these three reagents, suggesting that at least part of these epitopes are of a glycolipid nature. These Gal alpha 1----3Gal epitopes may thus serve as an antigenic source for the excess production of anti-Gal. In view of the naturally high level of anti-Gal in humans and its binding to T. cruzi and Leishmania, it is argued that these antibodies may contribute to the natural defense against the invasion of such parasites.     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Cysteine peptidases from Phytomonas serpens: biochemical and immunological approaches

Phytomonas serpens , a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi . In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens , while a strong 50-kDa cysteine peptidase was observed in T. cruzi . Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens . The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens , including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection.     

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.     

Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.     

Poly-N-acetyllactosamines synthesized by cultured Ehrlich carcinoma cells: application of endo-beta-galactosidase C for analysis of the terminal structure

Poly-N-acetyllactosamines were prepared from Ehrlich carcinoma cells cultured in the presence of [14C]galactose. Methylation analysis indicated that 31% of the galactose was in the non-reducing end. Of it, 77% was cleaved by alpha-galactosidase, and 56% was released as a disaccharide by endo-beta-galactosidase C. Methylation analysis confirmed that the released disaccharide was mostly Gal alpha 1----3Gal. Therefore, Gal alpha 1----3Gal structure, not Gal alpha 1----3(Gal alpha 1----6)Gal structure, was the major alpha-galactosyl structure in the poly-N-acetyllactosamines synthesized. Furthermore, alpha-galactosidase digestion did not change the content of disubstituted galactosyl residues. Thus, Gal alpha 1----3(Gal alpha 1----6)Gal structure, which was suggested to be the sole non-reducing terminal structure of poly-N-acetyllactosamines of Ehrlich carcinoma cells, was not detected in significant amounts under the present experimental conditions.     

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.     

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.     

Substrate specificity of the Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi trypomastigotes acquire sialic acid (SA) from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (TS). Here we study the substrate specificity of TS, which differs from all known sialyltransferases in that it does not require cytidine monophosphate (CMP)-SA as donor. The T. cruzi TS reversibly transfers SA to saccharides with terminal beta-Gal (but not alpha-Gal) residues. Donors are saccharides with SA linked to terminal beta-Gal residues by (alpha 2-3), but not (alpha 2-6) bonds. The type of beta-linkage of the terminal Gal residue is of minor importance (beta 1-4 and beta 1-6 are slightly better than beta 1-3), whereas chain length and the structure of additional vicinal sugar residues are not relevant. SA on the surface of living trypomastigotes of T. cruzi is transferred back and forth between the parasite surface and acceptor molecules with terminal beta-Gal, either in solution or on the surface of neighbouring mammalian cells. Addition of fucose residue on or close to the terminal galactose impairs TS activity. As a consequence, the enzyme acts poorly on the E-selectin ligand sialyl-Lewisx and its precursor Lewisx, and in vitro adhesion of TS-treated neutrophils to L-cells expressing L-selectin is not affected. Modifications in the structure of the (alpha 2-3)-linked N-acetyl-neuraminic acid (Neu5Ac) (deoxy or methoxy) of the donor molecules do not impair transfer if the changes are at C9, whereas changes at C4, C7 and C8 impair the ability to donate the modified SA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of carbohydrates recognized by the lectins Euonymus europaeus and concanavalin A in monoxenic and heteroxenic trypanosomatids.

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.     

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.     

Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids

Trypanosoma cruzi lipids contain a high content of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2). Previous data suggest that this parasite is able to convert oleic acid into linoleic acid; humans are not able to do this. Presently, we show that T. cruzi has a gene with high similarity to the delta12 (omega6)-oleate desaturase from plants. Northern blot analysis of the oleate desaturase gene from T. cruzi (OD(Tc)) indicated that this gene is transcribed in epimastigote, amastigote, and trypomastigote forms. Pulsed-field analysis showed that OD(Tc) is located at distinct chromosomal bands on distinct T. cruzi phylogenetic groups. In addition, the chromoblot analysis demonstrated the presence of homologous OD(Tc) genes in several trypanosomatids; namely, Crithidia fasciculata, Herpetomonas megaseliae, Leptomonas seymouri, Trypanosoma freitasi, Trypanosoma rangeli, Trypanosoma lewisi, Blastocrithidia sp., Leishmania amazonensis, Endotrypanum schaudinni, and Trypanosoma conorhini. The native OD(Tc) activity was detected by metabolic labeling and analysis of total fatty acids from epimastigotes and trypomastigotes of T. cruzi, coanomastigotes of C. fasciculata, and promastigotes of L. amazonensis, H. megaseliae, and L. seymouri. The fact that the enzyme oleate desaturase is not present in humans makes it an ideal molecular target for the development of new chemotherapeutic approaches against Chagas disease.     

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.     

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.     

Cationic amino acid uptake constitutes a metabolic regulation mechanism and occurs in the flagellar pocket of Trypanosoma cruzi

Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the possibility of using these molecules as a route of entry of therapeutic drugs.     

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.     

Arginine kinase: a common feature for management of energy reserves in African and American flagellated trypanosomatids

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.     

Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.