The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.     

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis.     

Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Monoclonal antibodies against Trypanosoma cruzi neuraminidase reveal enzyme polymorphism, recognize a subset of trypomastigotes, and enhance infection in vitro

The identification and characterization of two murine mAb (TCN-1 and TCN-2) that react with the neuraminidase of Trypanosoma cruzi is reported. The mAb were identified based on their ability to inhibit enzyme activity and recognize neuraminidase in crude enzyme preparations. TCN-1 and TCN-2 recognized Ag in tissue culture trypomastigotes but not in the amastigotes, epimastigotes, or metacyclic trypomastigotes using immunoblot assays and immunofluorescence. In addition, clones Y-H6, MV-13, and Silvio X-10/4 of T. cruzi revealed a unique banding pattern characteristic of each clone. In Silvio X-10/4, the mAb recognized four distinct bands ranging from 121,000 to 203,000 whereas in Y-H6 and MV-13 they identified bands ranging from 138,000 to 222,000. Characterization of neuraminidase by two-dimensional PAGE revealed the polypeptides that make up the enzyme to have isoelectrical points ranging from 6.55 to 7.30. Immunofluorescence and C-mediated lysis assays showed that the mAb reacted with a subset of trypomastigotes representing 28% of the total parasite population. Functional studies showed that the mAb enhanced infection of cultured cells by trypomastigotes. Our experiments confirm previous findings with polyclonal Ab and are in accordance with the hypothesis that neuraminidase modulates infection through a negative control mechanism.     

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.     

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.     

Heterologous expression of a Trypanosoma cruzi surface glycoprotein (gp82) in mammalian cells indicates the existence of different signal sequence requirements and processing

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.     

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.     

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.     

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.     

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation

Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.     

First report on Neotoma micropus (Rodentia) as a reservoir of Trypanosoma cruzi in Mexico]

Using xenodiagnosis, two (8.0%) of 25 woodrats Neotoma micropus were found infected with tripanosome parasite in Vaquerias, a village in Nuevo Leon State, Mexico. The triatomine species developing infective metacyclic trypanosomes at week 12th were Triatoma pallidipennis, T. infestans and T. gerstaeckeri. Experimental infections using infected dejections were successfully conducted on laboratory mice (CD1 strain) confirming the vertebrate cycle of Trypanosome cruzi. The biological characterization of T. cruzi strains was demonstrated based on: 1) Triatomine developmental cycle. 2) A vertebrate host parasitic period up to 25-33 post-infection days, and. 3) Typical morphology of bloodstream trypomastigotes and amastigotes from myocardial nest. This is the first report of T. cruzi biologically characterized in Nuevo Leon, as well as a new report of N. micropus, increasing the list of reservoir hosts in Mexico.     

In vivo and in vitro metacyclogenesis tests of two strains of Trypanosoma cruzi in the triatomine vectors Triatoma pseudomaculata and Rhodnius neglectus: short/long-term and comparative study

Metacyclogenesis of Trypanosoma cruzi of the Y and Berenice strains was studied in Triatoma pseudomaculata and Rhodnius neglectus. Results in vivo showed a higher production of metacyclic trypomastigotes in R. neglectus' digestive tube than in T. pseudomaculata. In vitro experiments were also carried out in order to compare the behavior of culture forms of T. cruzi incubated in extracts of different compartments (stomach, intestine, and rectum) of the digestive tract of both species of triatomines. A higher percentage of metacyclic trypomastigotes for both parasite strains, Y and Berenice, was detected in the rectum extract of R. neglectus in comparison to that from T. pseudomaculata. The same results were obtained with in vitro experiments, using parasites incubated in urine from each of those vectors. The adhesion of parasites to the incubated rectum epithelial cells was also compared. In incubations with the Y strain no significant differences were detected between the two triatomine species but, however, with the Berenice strain the mean percentage of cells with adhered parasites was higher in R. neglectus than in T. pseudomaculata.     

TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization

Here we have investigated the function of TcRho1, a Rho family orthologue from the parasite Trypanosoma cruzi. We have selected parasites overexpressing wild-type TcRho1 and a truncated form of TcRho1 (TcRho1-DeltaCaaX) which is unable to undergo farnesylation and supposed to interfere with recruitment of Rho effectors to membranes. TcRho1 protein was localized at the anterior region of wild-type and TcRho1 overexpressing epimastigotes, suggesting association with the Golgi apparatus. Accordingly, parasites overexpressing TcRho1-DeltaCaaX presented cytoplasmic fluorescence. To address the function of TcRho1 during differentiation, from epimastigotes to trypomastigotes, we submitted parasites overexpressing the above-cited lineages to metacyclogenesis assays. Parasites overexpressing TcRho1-DeltaCaaX generated a discrete number of metacyclic trypomastigotes when compared with other lineages. Strikingly, TcRho1-DeltaCaaX cells died synchronously during the process of metacyclogenesis.     

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.     

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.