RESUMEN

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.

RESUMEN

Endocytosis in Trypanosoma cruzi is mainly performed through a specialised membrane domain called cytostome-cytopharynx complex. Its ultrastructure and dynamics in endocytosis are well characterized in epimastigotes, being absent in trypomastigotes, that lack endocytic activity. Intracellular amastigotes also possess a cytostome-cytopharynx but participation in endocytosis of these forms is not clear. Extracellular amastigotes can be obtained from the supernatant of infected cells or in vitro amastigogenesis. These amastigotes share biochemical and morphological features with intracellular amastigotes but retain trypomastigote's ability to establish infection. We analysed and compared the ultrastructure of the cytostome-cytopharynx complex of intracellular amastigotes and extracellular amastigotes using high-resolution tridimensional electron microscopy techniques. We compared the endocytic ability of intracellular amastigotes, obtained through host cell lysis, with that of extracellular amastigotes. Intracellular amastigotes showed a cytostome-cytopharynx complex similar to epimastigotes'. However, after isolation, the complex undergoes ultrastructural modifications that progressively took to an impairment of endocytosis. Extracellular amastigotes do not possess a cytostome-cytopharynx complex nor the ability to endocytose. Those observations highlight morpho functional differences between intra and extracellular amastigotes regarding an important structure related to cell metabolism. TAKE AWAYS: T. cruzi intracellular amastigotes endocytose through the cytostome-cytopharynx complex. The cytostome-cytopharynx complex of intracellular amastigotes is ultrastructurally similar to the epimastigote. Intracellular amastigotes, once outside the host cell, disassembles the cytostome-cytopharynx membrane domain. Extracellular amastigotes do not possess a cytostome-cytopharynx either the ability to endocytose.

Asunto(s)

Enfermedad de Chagas , Trypanosoma cruzi , Membrana Celular , Endocitosis , Humanos , Microscopía Electrónica

RESUMEN

The parasite Trypanosoma cruzi causes Chagas' disease; both heme and ionic Fe are required for its optimal growth, differentiation, and invasion. Fe is an essential cofactor in many metabolic pathways. Fe is also harmful due to catalyzing the formation of reactive O2 species; for this reason, all living systems develop mechanisms to control the uptake, metabolism, and storage of Fe. However, there is limited information available on Fe uptake by T. cruzi. Here, we identified a putative 39-kDa Fe transporter in T. cruzi genome, TcIT, homologous to the Fe transporter in Leishmania amazonensis and Arabidopsis thaliana. Epimastigotes grown in Fe-depleted medium have increased TcIT transcription compared with controls grown in regular medium. Intracellular Fe concentration in cells maintained in Fe-depleted medium is lower than in controls, and there is a lower O2 consumption. Epimastigotes overexpressing TcIT, which was encountered in the parasite plasma membrane, have high intracellular Fe content, high O2 consumption-especially in phosphorylating conditions, high intracellular ATP, very high H2O2 production, and stimulated transition to trypomastigotes. The investigation of the mechanisms of Fe transport at the cellular and molecular levels will assist in elucidating Fe metabolism in T. cruzi and the involvement of its transport in the differentiation from epimastigotes to trypomastigotes, virulence, and maintenance/progression of the infection.

Asunto(s)

Trypanosoma cruzi , Metabolismo Energético , Homeostasis , Peróxido de Hidrógeno , Hierro , Estrés Oxidativo

RESUMEN

Significant advances have occurred in the area of high-resolution scanning electron microscopy (SEM), especially related to methodologies that allow the observation of intracellular structures that are exposed either by successive abrasion with a gallium ion beam or by sectioning in epoxy-embedded cells. Images of series of successively exposed surfaces can then be rendered into 3D models. Here, we report our observations by combining this approach with classical cytochemical methods to facilitate the 3D reconstruction of labeled structures and organelles. We used epimastigotes of Trypanosoma cruzi whose endocytic pathway was labeled with horseradish peroxidase, followed by fixation and detection of the peroxidase activity using the classical diaminobenzidine-osmium method followed by incubation with thiocarbohydrazide, which increases the concentration of osmium at the sites where the enzyme is located as well as the contrast of lipid-containing structures. This procedure allows not only a better visualization of membranous structures and lipid inclusions but can also easily identify the endocytic tracer (HRP) inside the cell. All structures involved in the endocytic activity could be traced and reconstructed.

Asunto(s)

Microscopía Electrónica de Rastreo , Trypanosoma cruzi/ultraestructura , Endocitosis , Histocitoquímica , Imagenología Tridimensional , Orgánulos/ultraestructura , Coloración y Etiquetado , Trypanosoma cruzi/metabolismo

RESUMEN

Trypanosoma cruzi epimastigote reservosomes store nutrients taken up during the intense endocytic activity exhibited by this developmental form. Reservosomes were classified as pre-lysosomal compartments. In contrast, trypomastigote forms are not able to take up nutrients from the medium. Interestingly, trypomastigotes also have acidic organelles with the same proteases contained in epimastigote reservosomes. Nevertheless, the origin and function of these organelles have not been disclosed so far. Given the similarities between the compartments of epimastigotes and trypomastigotes, the present study aimed to investigate the origin of metacyclic trypomastigote protease-containing organelles by tracking fluorospheres or colloidal gold particles previously stored in epimastigotes' reservosomes throughout metacyclogenesis. Using three-dimensional reconstruction of serial electron microscopy images, it was possible to find trypomastigote compartments containing the tracer. Our observations demonstrate that the protease-containing compartments from metacyclic trypomastigotes may originate directly from the reservosomes of epimastigotes.

Asunto(s)

Lisosomas/metabolismo , Trypanosoma cruzi/ultraestructura , Análisis de Varianza , Endocitosis/fisiología , Citometría de Flujo , Oro/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional , Estadios del Ciclo de Vida , Lisosomas/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo

RESUMEN

The cytostome-cytopharynx complex is the main site for endocytosis in epimastigotes of Trypanosoma cruzi It consists of an opening at the plasma membrane surface - the cytostome - followed by a membrane invagination - the cytopharynx. In G1/S cells, this structure is associated with two specific sets of microtubules, a quartet and a triplet. Here, we used electron microscopy and electron tomography to build 3D models of the complex at different stages of the cell cycle. The cytostome-cytopharynx is absent in late G2 and M phase cells, whereas early G2 cells have either a short cytopharynx or no visible complex, with numerous vesicles aligned to the cytostome-cytopharynx microtubules. The microtubule quartet remains visible throughout cell division (albeit in a shorter form), and is duplicated during G2/M. In contrast, the microtubule triplet is absent during late G2/M. Cells in cytokinesis have an invagination of the flagellar pocket membrane likely to represent early stages in cytostome-cytopharynx assembly. Cells in late cytokinesis have two fully developed cytostome-cytopharynx complexes. Our data suggest that the microtubule quartet serves as a guide for new cytostome-cytopharynx assembly.

Asunto(s)

División Celular , Estadios del Ciclo de Vida , Trypanosoma cruzi/citología , Trypanosoma cruzi/crecimiento & desarrollo , Citocinesis , Flagelos/metabolismo , Flagelos/ultraestructura , Fase G2 , Metafase , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Biológicos , Trypanosoma cruzi/ultraestructura

RESUMEN

Trypanosoma cruzi epimastigotes uptake nutrients by endocytosis via the cytostome-cytopharynx complex - an anterior opening (cytostome) continuous with a funnel-shaped invagination (cytopharynx) that extends to the posterior of the cell, accompanied by microtubules. During metacyclogenesis - the transformation of epimastigotes into human-infective metacyclic trypomastigotes - the cytostome-cytopharynx complex disappears, as trypomastigotes lose endocytic ability. To date, no studies have examined cytostome-cytopharynx complex disappearance in detail, or determined if endocytic activity persists during metacyclogenesis. Here, we produced 3D reconstructions of metacyclogenesis intermediates (Ia, Ib, Ic) using electron microscopy tomography and focused ion beam-scanning electron microscopy (FIB-SEM), concentrating on the cytostome-cytopharynx complex and adjacent structures, including the preoral ridge (POR). Parasite endocytic potential was examined by incubation of intermediate forms with the endocytic tracer transferrin (Tf)-Au. Ia, Ib and Ic cells were capable of internalizing Tf-Au, and had a shorter cytopharynx than that of epimastigotes, with the cytostome/POR progressively displaced towards the posterior, following the movement of the kinetoplast/flagellar pocket. While some Ic cells had a short cytopharynx with an enlarged proximal end (∼300nm in diameter, larger than that of the cytostome), other Ic cells had no cytopharynx invagination, but retained the cytopharynx microtubules, which were also present in metacyclics. We conclude that cytostome-cytopharynx disappearance and loss of endocytic ability are late events in metacyclogenesis, during which the cytostome is displaced towards the posterior, probably due to a link to the kinetoplast/flagellar pocket. Retention of the cytopharynx microtubules by metacyclics may allow prompt cytostome-cytopharynx reassembly in amastigotes, upon host cell infection.

Asunto(s)

Membrana Celular/química , Microtúbulos/química , Transferrina/química , Trypanosoma cruzi/química , Animales , Membrana Celular/ultraestructura , Tomografía con Microscopio Electrónico , Endocitosis/genética , Humanos , Microtúbulos/ultraestructura , Transferrina/ultraestructura , Trypanosoma cruzi/patogenicidad

RESUMEN

Insect trypanosomatids are inhabitants of the insect digestive tract. These parasites can be either monoxenous or dixenous. Plant trypanosomatids are known as insect trypanosomatids once they and are transmitted by phytophagous insects. Such parasites can be found in latex, phloem, fruits and seeds of many plant families. Infections caused by these pathogens are a major cause of serious economic losses. Studies by independent groups have demonstrated the metabolic flow of lipids from the vertebrate host to trypanosomatids. This mechanism is usually present when parasites possess an incomplete de novo lipid biosynthesis pathway. Here, we show that both insect trypanosomatids Phytomonas françai and Leptomonas wallacei incorporate (3)H-palmitic acid and inorganic phosphate. These molecules are used for lipid biosynthesis. Moreover, we have isolated the main hemolymphatic lipoprotein, Lipophorin (Lp) from Oncopeltus fasciatus, the natural insect vector of such parasites. Both parasites were able to incorporate Lp to be utilized both as a lipid and protein source for their metabolism. Also, we have observed the presence of Lp binding sites in the membrane of a parasite. In conclusion, we believe that the elucidation of trypanosomatid metabolic pathways will lead to a better understanding of parasite-host interactions and the identification of novel potential chemotherapy targets.

Asunto(s)

Interacciones Huésped-Parásitos , Metabolismo de los Lípidos , Lipoproteínas/metabolismo , Trypanosomatina/metabolismo , Animales , Sitios de Unión , Membrana Celular/metabolismo , Insectos/química , Insectos/parasitología , Lipoproteínas/aislamiento & purificación , Ácido Palmítico/metabolismo , Fosfatos/metabolismo

RESUMEN

The cytostome-cytopharynx complex is the main site of endocytosis of Trypanosoma cruzi epimastigotes. Little is known about the detailed morphology of this remarkable structure. We used serial electron tomography and focused-ion-beam scanning electron microscopy to reconstruct the entire complex, including the surrounding cytoskeleton and vesicles. Focusing on cells that had taken up gold-labeled tracers, we produced three-dimensional snapshots of the process of endocytosis. The cytostome cytoskeleton was composed of two microtubule sets--a triplet that started underneath the cytostome membrane, and a quartet that originated underneath the flagellar-pocket membrane and followed the preoral ridge before reaching the cytopharynx. The two sets accompanying the cytopharynx formed a 'gutter' and left a microtubule-free side, where vesicles were found to be associated. Cargo was unevenly distributed along the lumen of the cytopharynx, forming clusters. The cytopharynx was slightly longer during the G2 phase of the cell cycle, although it did not reach the postnuclear region owing to a bend in its path. Therefore, the cytopharynx is a dynamic structure, undergoing remodeling that is likely associated with endocytic activity and the preparation for cell division.

Asunto(s)

Trypanosoma cruzi/ultraestructura , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Tomografía con Microscopio Electrónico/métodos , Endocitosis , Microtúbulos/metabolismo , Trypanosoma cruzi/metabolismo

RESUMEN

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-ß-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

Asunto(s)

Endocitosis/fisiología , Leishmania mexicana/metabolismo , Lipoproteínas LDL/metabolismo , Microdominios de Membrana/metabolismo , Receptores de LDL/metabolismo , Animales , Bovinos , Ésteres del Colesterol/metabolismo , Esterificación , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/crecimiento & desarrollo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangre , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , beta-Ciclodextrinas/farmacología

RESUMEN

Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.

Asunto(s)

Microcuerpos/metabolismo , Fosfoglucomutasa/química , Fosfoglucomutasa/metabolismo , Señales de Clasificación de Proteína , Trypanosoma cruzi/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Fosfoglucomutasa/genética , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Distribución Tisular , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo

RESUMEN

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

Asunto(s)

Endocitosis , Eucariontes , Animales , Entamoeba/metabolismo , Entamoeba/fisiología , Entamoeba/ultraestructura , Eucariontes/metabolismo , Eucariontes/fisiología , Eucariontes/ultraestructura , Giardia/metabolismo , Giardia/fisiología , Giardia/ultraestructura , Histocitoquímica , Leishmania/metabolismo , Leishmania/fisiología , Leishmania/ultraestructura , Microscopía Electrónica , Plasmodium/metabolismo , Plasmodium/fisiología , Plasmodium/ultraestructura , Trichomonas/metabolismo , Trichomonas/fisiología , Trichomonas/ultraestructura , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiología , Trypanosoma brucei brucei/ultraestructura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/ultraestructura

RESUMEN

Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.

Asunto(s)

Cromatografía Liquida , Vesículas Citoplasmáticas/química , Espectrometría de Masas , Proteínas Protozoarias/análisis , Fracciones Subcelulares/química , Trypanosoma cruzi/química , Animales , Fraccionamiento Celular , Vesículas Citoplasmáticas/ultraestructura , Metabolismo de los Lípidos , Microscopía Electrónica de Transmisión , Proteómica/métodos , Fracciones Subcelulares/ultraestructura , Trypanosoma cruzi/ultraestructura

RESUMEN

Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions.

Asunto(s)

Endosomas/ultraestructura , Trypanosoma cruzi/ultraestructura , Animales , Microscopía por Crioelectrón , Membranas Intracelulares/ultraestructura , Microscopía Electrónica de Transmisión , Microtomía , Compuestos Organometálicos , Coloración y Etiquetado

RESUMEN

Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of researchers. These studies have provided new information of the cell biology of these parasites and have supported investigators to assume that some of the protozoa form the roots of the evolutionary tree of eukaryotic cells. The cell fractionation usually starts with disruption of the plasma membrane, using conditions that minimize damage to the membranes bounding intracellular organelles. An important requirement for successful cell fractionation is the evaluation of the isolation procedure that can be made by morphological and biochemical methods. The morphological approaches use light and electron microscopy of thin section of different fractions obtained, and the biochemical methods are based on the quantification of marker enzymes or other molecules (for instance, a special type of lipid, an antigen, etc.). Here we will present our experience in the isolation and characterization of some structures found in trypanosomatids and trichomonads.

Asunto(s)

Fraccionamiento Celular , Eucariontes/citología , Animales , Electroforesis en Gel de Poliacrilamida , Eucariontes/ultraestructura , Microscopía Electrónica

RESUMEN

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.

Asunto(s)

Macrófagos/parasitología , Factor de Activación Plaquetaria/análisis , Proteínas Protozoarias/análisis , Trypanosoma cruzi/química , Animales , Western Blotting/métodos , Técnica del Anticuerpo Fluorescente , Estadios del Ciclo de Vida , Ratones , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria , Proteínas Protozoarias/farmacología , Conejos , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and beta-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable ( approximately 50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.

Asunto(s)

Cisteína Endopeptidasas/metabolismo , Aparato de Golgi/parasitología , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidad , Animales , Western Blotting , Calreticulina/metabolismo , Diferenciación Celular , Microscopía por Crioelectrón , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/farmacología , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Aparato de Golgi/metabolismo , Concentración 50 Inhibidora , Hígado/metabolismo , Lisosomas/metabolismo , Mercaptoetanol/farmacología , Microscopía Fluorescente , Octoxinol/farmacología , Orgánulos/metabolismo , Fenotipo , Unión Proteica , Proteínas Recombinantes/química , Dodecil Sulfato de Sodio/química , Dodecil Sulfato de Sodio/farmacología , Factores de Tiempo , Transfección

RESUMEN

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.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Estadios del Ciclo de Vida , Proteínas Protozoarias/metabolismo , Fracciones Subcelulares/enzimología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Proteínas de Unión al GTP rho/metabolismo , Animales , Apoptosis/fisiología , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Distribución Tisular , Trypanosoma cruzi/citología

RESUMEN

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.

Asunto(s)

Aparato de Golgi/metabolismo , Trypanosoma cruzi/citología , Trypanosoma cruzi/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Sueros Inmunes/inmunología , Transporte de Proteínas , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7

RESUMEN

During its life cycle Trypanosoma rangeli crosses the hemolymph of its invertebrate host. In the present study, we demonstrate for the first time the uptake of lipophorin (Lp), the main lipid-transporting particle of insect hemolymph. We observed that living T. rangeli parasites uptake lipids from both 32P- and 3H-, or 125I-labeled Lp. However, the parasites do not uptake any other hemolymphatic protein such as 32P-labeled vitellogenin. The presence of a specific receptor to Lp in the parasite surface is suggested based on experiments using 125I-Lp. We also investigated the intracellular fate of lipids using Texas Red-labeled phosphatidylethanolamine-Lp. Parasites were observed under confocal microscope and displayed fluorescent-labeled lipids close to the flagellar pocket and in vesicles at the posterior region. In conclusion, this study raises a novel set of molecular events which takes place during vector-parasite interaction.

Asunto(s)

Proteínas Portadoras/metabolismo , Insectos Vectores/metabolismo , Lipoproteínas/metabolismo , Rhodnius/metabolismo , Trypanosoma/metabolismo , Animales , Proteínas Portadoras/sangre , Endocitosis , Hemolinfa/metabolismo , Interacciones Huésped-Parásitos , Cinética , Metabolismo de los Lípidos , Lipoproteínas/sangre , Rhodnius/parasitología , Trypanosoma/citología , Trypanosoma/fisiología