Trypomastigote Differentiation Research Articles

Intracellular DNA replication and differentiation of Trypanosoma cruzi is asynchronous within individual host cells in vivo at all stages of infection.

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.

Redox Paradox: The Contribution of Reactive Oxygen Species (ROS) in the Proliferation and Differentiation of Trypanosoma cruzi

Trypanosoma cruzi is a protozoan that causes Chagas disease or American trypanosomiasis. The parasite has a heteroxenic biological cycle developing between a vertebrate host (mammal) and an invertebrate (insect vector). The first environment encountered by T. cruzi after the blood meal is the midgut of the insect, where large amounts of hemoglobin are degraded resulting in the release of huge concentrations of heme, a molecule known to increase the formation of ROS. Heme induces T. cruzi proliferation, and this phenomenon is accompanied by a marked time and concentration dependent increase in ROS formation and the modulation of epimastigotes mitochondrial physiology in an effort to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation. Conversely, the antioxidants reverse the heme-induced ROS and dramatically impair epimastigotes proliferation. Following the cycle, when these forms are taken into the hindgut of the triatomine they return to the non-replicative, infective form, the metacyclic trypomastigotes. During the metacyclogenesis, antioxidants induce a significant increment of trypomastigotes. These data gives us the idea of a “shift” between proliferation to differentiation according to the influence of the redox status. Surprisingly, when metacyclic trypomastigotes are released into the insect feces the infection is again favored by ROS since inhibitors of Nox and antioxidants greatly decrease macrophage infection. Moreover, while the parasite needs oxidants for its proliferation inside the vector, the opposite occurs during its development inside the vertebrate host. H2O2 diminishes amastigotes proliferation and increases the trypomastigote differentiation into amastigotes. Taken together, our data reveal a redox paradox in which ROS plays antagonistic roles along Trypanosoma cruzi life cycle demonstrating a high capacity to adapt to its habitats.

SFRR-E Young Investigator Awardee P85 - Nitric oxide affects trypomastigote to amastigote differentiation in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas disease. Two main distinct forms are present in the mammalian host: trypomastigote, an infective form, and the amastigote, a typical replicative form. Succeeding the host cell invasion, trypomastigotes differentiate to amastigotes, a process known as amastigogenesis. Amastigogenesis is characterized by parasite body remodeling, with drastic reduction of flagellum, and changes in protein profile and energetic metabolism. Our aim is to explore the role of nitric oxide as a signaling molecule during the amastigogenesis process, which must be strictly regulated. We report herein that acid pH (6.0) is essential for T. cruzi amastigogenesis. Also, during amastigogenesis there is a progressive solubilization of the paraflagellar protein, a flagellum marker. Moreover, the process is dependent on •NO concentration, since it is suppressed by 1mM SNAP, a •NO donor, and favored by 10mM L-NAME, a NOS inhibitor. Accordingly, S-nitrosylation of selective proteins occurs in amastigogenesis. Additionally, amastigogenesis is affected by IBMX (PDE inhibitor) treatment, suggesting the importance of cyclic nucleotides signaling. Furthermore, tubulin stability is also affected by the •NO availability. Along amastigogenesis, flagellum disassembling is accompanied by changes in a-tubulin tyrosylation and polyglutamylation levels. Taken together, these results suggest •NO participation in trypomastigote differentiation to amastigotes in T. cruzi.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

Inositolphosphoceramide Metabolism in Trypanosoma cruzi as Compared with other Trypanosomatids

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.

Developmental Expression of a Trypanosoma cruzi Phosphoinositide-Specific Phospholipase C in Amastigotes and Stimulation of Host Phosphoinositide Hydrolysis

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus and localizes to the outer surface of the plasma membrane of amastigotes. We show here that TcPI-PLC is developmentally regulated in amastigotes and shows two peaks of surface expression during the developmental cycle of T. cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the second before differentiation of amastigotes into trypomastigotes. Surface expression of TcPI-PLC coincides with phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion in the host cell membrane and with an increase in the levels of its product, inositol 1,4,5-trisphosphate. During extracellular differentiation, PI-PLC is secreted into the incubation medium. Maximal early expression of TcPI-PLC on the surface of amastigotes and PIP(2) depletion coincide with host cytoskeletal changes, Ca(2+) signaling, and transcriptional responses described previously. The presence of TcPI-PLC on the outer surface of the plasma membrane of the parasite and the capacity to be secreted and to alter host phospholipids are novel mechanisms of the host-parasite interaction.

Exploring Signaling Events Surrounding Extracellular Amastigote Invasion Processes Of Trypanosoma Cruzi

Trypanosoma cruzi is a protozoan pathogen that infects humans and other mammals, producing a pathology called Chagas disease. The disease is endemic in most of central and South America affecting 18 million people, with an increasing number of cases in North America. Amastigotes which are generated by the extracellular differentiation of trypomastigotes are referred to as Extracellular Amastigotes (EA) and are able to invade cultured cells. EA is dependent on host actin filaments polymerization to invade cells. Cortactin has emerged as a key signaling protein in cellular processes such as endocytosis and tumor invasion. The ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Cortactin showed to be a substrate of PKD phosphorylation in vivo. In this study, cells were transfected with cortactin and PKD‐GFP‐vectors, infected with EA and examined for the acquisition of these markers. Cells were previously treated with kinase (Fyn‐Src, PI3K) inhibitors and control cells were left untreated. PKD is recruited to the sites of actin remodeling during EA invasion, which also recruits cortactin. Co‐localization of PKD and cortactin may be an indication that PKD plays a role in cytoskeletal reorganization. The inhibition of PI3K pathway enhances both EA invasion and LAMP‐1 recruitment. Here, we propose a signaling pathway model of EA entry.

Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast -- an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.

SB-431542, a Transforming Growth Factor β Inhibitor, ImpairsTrypanosoma cruziInfection in Cardiomyocytes and Parasite Cycle Completion

The antiinflammatory cytokine transforming growth factor beta (TGF-beta) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-beta type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-beta pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-beta signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-beta signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.

A Lipid-modified Phosphoinositide-specific Phospholipase C (TcPI-PLC) Is Involved in Differentiation of Trypomastigotes to Amastigotes of Trypanosoma cruzi

The phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the inositol phosphate/diacylglycerol signaling pathway. A newly discovered Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus, targeted to its plasma membrane, and believed to play a role in differentiation of the parasite because its expression increases during the differentiation of trypomastigote to amastigote stages. To determine whether TcPI-PLC is involved in this differentiation step, antisense inhibition using phosphorothioate-modified oligonucleotides, and overexpression of the gene were performed. Antisense oligonucleotide-treated parasites showed a reduced rate of differentiation in comparison to controls, as well as accumulation of intermediate forms. Overexpression of TcPI-PLC led to a faster differentiation rate. In contrast, overexpression of a mutant TcPI-PLC that lacked the lipid modification at its N terminus did not affect the differentiation rate. Therefore, TcPI-PLC is involved, when expressed in the plasma membrane, in the differentiation of trypomastigotes to amastigotes, an essential step for the intracellular replication of these parasites.

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.

Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi

We present the results of the first detailed study of the molecular and cellular basis of the antiproliferative effects of the bisphosphonate risedronate (Ris) on Trypanosoma cruzi, the causative agent of Chagas’ disease. Ris and related compounds, which block poly-isoprenoid biosynthesis at the level of farnesyl pyrophosphate synthase, are currently used for the treatment of bone resorption disorders, but also display selective activity against trypanosomatid and apicomplexan parasites. Ris induced a dose-dependent effect on growth of the extracellular epimastigote form of T. cruzi; complete growth arrest and cell lysis ensued at 150 μM. Growth inhibition was associated with depletion of the parasite’s endogenous sterols, but complete growth arrest and loss of cell viability took place before full depletion of these compounds, suggesting that disappearance of other essential poly-isoprenoids is involved in its anti-parasitic action. Ris had a variety of effects on cellular ultrastructure, including mitochondrial swelling, disorganisation of other organelles, such as reservosomes and the kinetoplast, together with the appearance of autophagic vesicles and progressive vacuolization of the cytoplasm. Ris had selective antiproliferative effects against the clinically relevant amastigote form of T. cruzi, and at 100 μM, was able to prevent completely the development of T. cruzi infection of murine muscle heart or Vero cells, and to cure cultures which were already infected. Ris induced drastic ultrastructural alterations in the intracellular parasites and blocked amastigote to trypomastigote differentiation, with no biochemical or ultrastructural effects on the host cells, which fully recovered their normal structure and activity after treatment. Ris is, therefore, a promising lead compound for the development of new drugs against T. cruzi.

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote.

Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [(3)H]palmitic acid or [(3)H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using (3)H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A(1), phospholipase A(2), inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

A Novel Phosphatidylinositol-Phospholipase C of Trypanosoma cruzi That Is Lipid Modified and Activated during Trypomastigote to Amastigote Differentiation

The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian delta-type PI-PLCs. The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity. In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its amino-terminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains. TcPI-PLC is lipid modified in vivo, as demonstrated by metabolic labeling with [(3)H]myristate and [(3)H]palmitate and fatty acid analysis of the immunoprecipitated protein, and may constitute the first example of a new group of PI-PLCs.

Features of host cell invasion by different infective forms of Trypanosoma cruzi.

Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.

Involvement of calyculin A-sensitive phosphatase(s) in the differentiation of Trypanosoma cruzi trypomastigotes to amastigotes

Differentiation of the non-dividing trypomastigote form of Trypanosoma cruzi, the causative agent of Chagas disease, to the dividing amastigote form normally occurs in cytoplasm of infected cells. Here we show that calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, induces at pH 7.5 extracellular transformation of long slender trypomastigotes to round amastigote-like forms which acquire characteristic features observed after the normal differentiation process: repositioning and structural changes of the kinetoplast, release of surface neuraminidase, and expression of amastigote-specific epitopes. Calyculin A inhibits parasite phosphatases and changes in the phosphorylation of specific proteins occur during the transformation process. As an exposure of trypomastigotes to calyculin A concentrations as low as 1 nM and for only 1–2 h is sufficient to induce transformation, the inhibition of calyculin A-sensitive phosphatase(s) appears to play a major role in initiating the trypomastigote differentiation.

Developmentally regulated expression of ceramide in Trypanosoma cruzi

Amastigote forms of T. cruzi express the specific Ssp-4 surface antigen which is progressively shed, by the action of an endogenous phosphatidylinositol-phospholipase C, during their development into epimastigotes (Andrews et al., J. Exp. Med., 167 (1988) 300–314). We show now that the lipid moiety of the anchor of Ssp-4 is a ceramide which was metabolically labelled with [ 3H]palmitic acid. The lipid could be cleaved by PI-PLC digestion in vitro, and was identified by methanolysis and reverse phase thin layer chromatography of the products, as palmitoyldihydrosphingosine. Also, the free biosynthesized lipids were investigated in parasites obtained after 0, 24, 48 and 72 h differentiation of trypomastigotes and further incubated with [ 3H]palmitic acid for 2 h. A maximum of free ceramide was found in the 24 h point, in accordance with the maximum of amastigote forms. In contrast only traces of free ceramide were found in trypomastigotes. The major ceramide (more than 90%) is palmitoyldihydrosphingosine, which is the same as found in the anchor of Ssp-4. The ceramide could play an important role in the cell biology of the parasite as previously found for mammalian cells.

Trypanosoma cruzi: Differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Specific inhibition of the morphogenesis of Trypanosoma cruzi by a monoclonal antibody.

Trypanosoma cruzi, the causative agent of Chagas' disease, is transmitted primarily by blood-sucking reduviid bugs. The insects themselves are infected by ingesting trypomastigotes with the blood of infected mammals. On ingestion by the vector, the parasites convert to epimastigotes which multiply within the lumen of the crop and midgut. In the hindgut and rectum of the reduviid, the epimastigotes transform into non-dividing metacyclic trypomastigotes which can, when passed with the faeces, infect the vertebrate host1. Lectins capable of binding T. cruzi have been isolated from different regions of the vector's intestinal tract2,3. It has been proposed that the interaction of these lectins with the surface membrane of the parasite regulates the morphogenesis of T. cruzi in the gut of the vector2,3. In the present study we describe the inhibition of epimastigote to trypomastigote differentiation in vitro by a monoclonal antibody specific for a 72,000 molecular weight cell-surface glyco-protein of T. cruzi and propose that this molecule may be a receptor controlling parasite differentiation in the vector.