Shotgun sequencing analysis of Trypanosoma cruzi I Sylvio X10/1 and comparison with T. cruzi VI CL Brener

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite.     

Identification of genes encoding hypothetical proteins in open-reading frame expressed sequence tags from mammalian stages of Trypanosoma cruzi

Approximately 50% of the predicted protein-coding genes of the Trypanosoma cruzi CL Brener strain are annotated as hypothetical or conserved hypothetical proteins. To further characterize these genes, we generated 1161 open-reading frame expressed sequence tags (ORESTES) from the mammalian stages of the VL10 human strain. Sequence clustering resulted in 435 clusters, consisting of 339 singletons and 96 contigs. Significant matches to the T. cruzi predicted gene database were found for ~94% contigs and ~69% singletons. These included genes encoding surface proteins, known to be intensely expressed in the parasite mammalian stages and implicated in host cell invasion and/or immune evasion mechanisms. Among 151 contigs and singletons with similarity to predicted hypothetical protein-coding genes and conserved hypothetical protein-coding genes, 83% showed no match with T. cruzi EST and/or proteome databases. These ORESTES are the first experimental evidence that the corresponding genes are in fact transcribed. Sequences with no significant match were searched against several T. cruzi and National Center for Biotechnology Information non-redundant sequence databases. The ORESTES analysis indicated that 124 predicted conserved hypothetical protein-coding genes and 27 predicted hypothetical protein-coding genes annotated in the CL Brener genome are transcribed in the VL10 mammalian stages. Six ORESTES annotated as hypothetical protein-coding genes showing no match to EST and/or proteome databases were confirmed by Northern blot in VL10. The generation of this set of ORESTES complements the T. cruzi genome annotation and suggests new stage-regulated genes encoding hypothetical proteins.     

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.     

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.     

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.     

The binding of CCL2 to the surface of Trypanosoma cruzi induces chemo-attraction and morphogenesis

Adhesion of Trypanosoma cruzi to host cells employs mechanisms which are complex and not completely understood. Upon infection, host cells release pro-inflammatory cytokines and chemokines in the environment. These had been found to be involved with increasing parasite uptake as well as killing by macrophages and cardiomyocytes. In the present study, we focused on the interaction of murine beta-chemokine CCL2 with trypomastigote forms of T. cruzi. We found that this chemokine directly triggers the chemotaxis and morphogenesis of trypomastigote forms of parasites. Binding assays showed that the interaction of CCL2 with molecules present in trypomastigote forms is abolished by the addition of condroitin 6-sulphate, a glycosaminoglycan. Moreover, we also observed that the parasite glycoproteins are the major players in this interaction. In summary, our study demonstrates a host ligand/parasite receptor interaction that may have relevant implications in the tissue tropism of this important parasitic disease.     

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 μM. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Trypanosoma cruzi: protection in mice immunized with 8-methoxypsoralen-inactivated trypomastigotes

Trypomastigote forms from the Y strain of Trypanosoma cruzi were inactivated by treatment with 8-methoxypsoralen and ultraviolet radiation (365 nm). The parasite population maintained normal morphology, mobility, and mammalian cell invasion capacity, being incapable of intracellular differentiation and reproduction. A strong protection of inbred A/Snell mice against challenges with virulent T. cruzi forms was obtained through three inoculations of the inactivated trypomastigotes. All immunized mice survived, with negative parasitemias and absence of tissue lesions. Several antibody-mediated reactions were performed with sera from the protected mice at distinct stages of the experiment. The levels of agglutinating, lytic (complement-mediated), and protein A binding antibodies increased progressively with each immunizing booster. The trypomastigote surface proteins recognized by antibodies present in these sera were identified after immunoprecipitation and two-dimensional polyacrylamide gel electrophoresis.     

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH(4)Cl, which releases Ca(2+) from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.     

Trypanosoma cruzi: surface charge and freeze-fracture of amastigotes

Amastigotes of Trypanosoma cruzi, within vertebrate cells or isolated from the supernatant of vertebrate cell cultures (L-A9 fibroblast or J774G8 macrophage-like cell lines), possess glycoproteins or glycolipids on the cell surface according to the periodic acid-thiosemicarbazide-silver proteinate technique used in association with electron microscopy. The cell surface of isolated amastigotes is negatively charged, as evaluated by the binding of cationic particles (colloidal iron hydroxyde at pH 1.8 and cationized ferritin at pH 7.2) as well as by direct measurement of cellular electrophoretic mobility. Amastigotes (Y strain) isolated from the spleen of infected mice and amastigotes (Y and CL strains) from the supernatant of cell cultures previously infected with T. cruzi have the same mean electrophoretic mobility (-0.85 micron sec-1 V-1 cm). It is intermediate between the epimastigote and the trypomastigote forms (determined previously). Sialic acid is the important component responsible for the negative surface charge, as determined by the use of neuraminidase. Thus, it is possible to use the mean electrophoretic mobility as an indicator for identifying amastigotes of T. cruzi.     

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.     

Fluorescence-activated cell-sorting analysis of fibronectin peptides binding to Trypanosoma cruzi trypomastigotes

The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells.     

Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells

The involvement of actin filaments from the host cell on the process of invasion of trypomastigote forms of Trypanosma cruzi was analyzed in seven different cell lines. Prior incubation of all cell lines with cytochalasin D, under conditions which interfere with actin filaments, markedly inhibited parasite internalization and increased parasite attachment. Attached parasites were readily ingested following washing of the drug-treated cells. Cytochalasin treatment interfered with the distribution of actin filaments of the host cell as evaluated by visualization of the filaments using confocal laser scanning microscopy of cells incubated in the presence of FITC-phalloidin. Concentration of actin filaments could be observed in most, but not all, parasites in the process of internalization. We also treated LLCMK 2 and macrophage cells with Jasplakinolide, a drug that stabilizes actin filaments, before interaction with the trypomastigote forms. This drug partially inhibits parasite invasion into the cells. Prior incubation of the host cells in the presence of colchicine, which interfere with microtubules, also inhibited parasite internalization into the cells.     

MRNA encoding a putative RNA helicase of the DEAD-box gene family is up-regulated in trypomastigotes of Trypanosoma cruzi

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.     

Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca(2+) response both in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T. cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase inhibitor genistein reduced by approximately 75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein tyrosine kinase mediated the phosphorylation of a 175kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores, or thapsigargin, an inhibitor of intracellular Ca(2+) transport ATPases, the infectivity of the CL but not of the G isolate diminished significantly (P<0.005). In both isolates, a combination of ionomycin plus NH(4)Cl or nigericin released Ca(2+) from acidic vacuoles containing a Ca(2+)/H(+) exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate. Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the two isolates, leading to Ca(2+) release from different intracellular compartments.