Iron Uptake Controls Trypanosoma cruzi Metabolic Shift and Cell Proliferation.

(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.

The cytostome-cytopharynx complex of intracellular and extracellular amastigotes of Trypanosoma cruzi exhibit structural and functional differences.

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.

An Iron Transporter Is Involved in Iron Homeostasis, Energy Metabolism, Oxidative Stress, and Metacyclogenesis in Trypanosoma cruzi.

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.

Trypanosomatid Flagellar Pocket from Structure to Function.

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are flagellate eukaryotic parasites that cause serious diseases in humans and animals. These parasites have cell shapes defined by a subpellicular microtubule array and all share a number of important cellular features. One of these is the flagellar pocket, an invagination of the cell membrane around the proximal end of the flagellum, which is an important organelle for endo/exocytosis. The flagellar pocket plays a crucial role in parasite pathogenicity and persistence in the host and has a great influence on cell morphogenesis and cell division. Here, we compare the morphology and function of the flagellar pockets between different trypanosomatids, with their life cycles and ecological niches likely influencing these differences.

Electron microscopy cytochemistry and three-dimensional reconstruction of labeled structures in Trypanosoma cruzi.

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.

Control of assembly of extra-axonemal structures: the paraflagellar rod of trypanosomes.

Eukaryotic flagella are complex microtubule-based organelles that, in many organisms, contain extra-axonemal structures, such as the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition zone and the flagellum itself. The process begins with the translation of protein components followed by their sorting and trafficking into the flagellum, transport to the assembly site and incorporation. Flagella are formed from over 500 proteins and the principles governing assembly of the axonemal components are relatively clear. However, the coordination and location of assembly of extra-axonemal structures are less clear. We have discovered two cytoplasmic proteins in Trypanosoma brucei that are required for PFR formation, PFR assembly factors 1 and 2 (PFR-AF1 and PFR-AF2, respectively). Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The existence of cytoplasmic factors required for PFR formation aligns with the concept that processes facilitating axoneme assembly occur across multiple compartments, and this is likely a common theme for extra-axonemal structure assembly.

Tridimensional Electron Microscopy Analysis of the Early Endosomes and Endocytic Traffic in Trypanosoma cruzi Epimastigotes.

Trypanosoma cruzi epimastigotes internalize macromolecules avidly by endocytosis. Previously, we identified a tubule-vesicular network likely to correspond to the early-endosomes. However, a detailed ultrastructural characterization of these endosomes was missing. Here, we combined endocytosis assays with ultrastructural data from high-resolution electron microscopy to produce a 3D analysis of epimastigote endosomes and their interactions with endocytic organelles. We showed that endocytic cargo was found in carrier vesicles budding from the cytopharynx. These vesicles appeared to fuse with a tubule-vesicular network of early endosomes identified by ultrastructural features including the presence of intermembrane invaginations and coated membrane sections. Within the posterior region of the cell, endosomes localized preferentially on the side nearest to the cytopharynx microtubules. At 4°C, cargo accumulated at a shortened cytopharynx, and subsequent temperature shift to 12°C led to slow cargo delivery to endosomes and, later, to reservosomes. Bridges between reservosomes and endosomes resemble heterotypic fusion. Reservosomes are excluded from the posterior end of the cell, with no preferential cargo delivery to reservosomes closer to the nucleus. Our 3D analysis indicates that epimastigotes accomplish high-speed endocytic traffic by cargo transfer to a bona fide early-endosome and then directly from endosomes to reservosomes, via multiple and simultaneous heterotypic fusion events.

Trypanosoma cruzi epimastigotes store cholesteryl esters in lipid droplets after cholesterol endocytosis.

The Chagas disease agent Trypanosoma cruzi proliferates in the insect vector as highly endocytic epimastigotes that store nutrients, including lipids in reservosomes (lysosome related compartments). Although nutrient storage is important for epimastigote transformation into infective metacyclics, the epimastigote lipid droplets (LDs) remain uncharacterized. Here, we characterized the epimastigote LDs and examined their relationship with the endocytic pathway. Fluorescence microscopy using BODIPY showed that LDs have high neutral lipid content and harbor Rab18, differently from other lipid-rich organelles (such as reservosomes). Using transmission electron microscopy (TEM), we observed a close relationship between LDs and the endoplasmic reticulum, mitochondria and glycosomes. We developed a reproducible protocol to isolate LDs, and showed (by HTPLC and GC/MS analyses) that they have 89% neutral lipids and 11% phospholipids, which are likely to form the LD monolayer seen by TEM. The LD neutral lipids were mostly sterols, although triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids (FFA) were also found. Endocytosis of 3H-labeled cholesterol-BSA showed that internalized cholesterol is stored in LDs mostly in the cholesteryl ester form. Together, these results suggest that exogenous cholesterol internalized by endocytosis reaches the reservosomes and is then stored into LDs after esterification.

Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-ß oligomers.

Alzheimer's disease (AD) is a disabling and highly prevalent neurodegenerative condition, for which there are no effective therapies. Soluble oligomers of the amyloid-ß peptide (AßOs) are thought to be proximal neurotoxins involved in early neuronal oxidative stress and synapse damage, ultimately leading to neurodegeneration and memory impairment in AD. The aim of the current study was to evaluate the neuroprotective potential of mesenchymal stem cells (MSCs) against the deleterious impact of AßOs on hippocampal neurons. To this end, we established transwell cocultures of rat hippocampal neurons and MSCs. We show that MSCs and MSC-derived extracellular vesicles protect neurons against AßO-induced oxidative stress and synapse damage, revealed by loss of pre- and postsynaptic markers. Protection by MSCs entails three complementary mechanisms: 1) internalization and degradation of AßOs; 2) release of extracellular vesicles containing active catalase; and 3) selective secretion of interleukin-6, interleukin-10, and vascular endothelial growth factor to the medium. Results support the notion that MSCs may represent a promising alternative for cell-based therapies in AD.

Lysosome-like compartments of Trypanosoma cruzi trypomastigotes may originate directly from epimastigote reservosomes.

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.

The cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes disassembles during cell division.

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.

Loss of the cytostome-cytopharynx and endocytic ability are late events in Trypanosoma cruzi metacyclogenesis.

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.

99m-Technetium binding site in bone marrow mononuclear cells.

INTRODUCTION: The increasing interest in 99m-technetium ((99m)Tc)-labeled stem cells encouraged us to study the (99m)Tc binding sites in stem cell compartments. METHODS: Bone marrow mononuclear cells were collected from femurs and tibia of rats. Cells were labeled with (99m)Tc by a direct method, in which reduced molecules react with (99m)Tc with the use of chelating agents, and lysed carefully in an ultrasonic apparatus. The organelles were separated by means of differential centrifugation. At the end of this procedure, supernatants and pellets were counted, and the percentages of radioactivity (in megabecquerels) bound to the different cellular fractions were determined. Percentages were calculated by dividing the radioactivity in each fraction by total radioactivity in the sample. The pellets were separated and characterized by their morphology on electron microscopy. RESULTS: The labeling procedure did not affect viability of bone marrow mononuclear cells. Radioactivity distributions in bone marrow mononuclear cell organelles, obtained in five independent experiments, were approximately 38.5 % in the nuclei-rich fraction, 5.3 % in the mitochondria-rich fraction, 2.2 % in microsomes, and 54 % in the cytosol. Our results showed that most of the radioactivity remained in the cytosol; therefore, this is an intracellular labeling procedure that has ribosomes unbound to membrane and soluble molecules as targets. However, approximately 39 % of the radioactivity remained bound to the nuclei-rich fraction. To confirm that cell disruption and organelle separation were efficient, transmission electron microscopy assays of all pellets were performed. CONCLUSIONS: Our results showed that most of the radioactivity was present in the cytosol fraction. More studies to elucidate the mechanisms involved in the cellular uptake of (99m)Tc in bone marrow cells are ongoing.

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions.

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.

Lipophorin Drives Lipid Incorporation and Metabolism in Insect Trypanosomatids.

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.

The three-dimensional structure of the cytostome-cytopharynx complex of Trypanosoma cruzi epimastigotes.

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.

Assembly of the Leishmania amazonensis flagellum during cell differentiation.

The flagellar cytoskeleton of Leishmania promastigotes contains the canonical 9+2 microtubular axoneme and a filamentous structure, the paraflagellar rod (PFR), which is present alongside the axoneme. In contrast to promastigotes, which contain a long and motile flagellum, the amastigote form of Leishmania displays a short flagellum without a PFR that is limited to the flagellar pocket domain. Here, we investigated the biogenesis of the Leishmania flagellum at 0, 4, 6 and 24h of differentiation. Light and electron microscopy observations of the early stages of L. amazonensis differentiation showed that the intermediate forms presented a short and wider flagellum that did not contain a PFR and presented reduced motion. 3D-reconstruction analysis of electron tomograms revealed the presence of vesicles and electron-dense aggregates at the tip of the short flagellum. In the course of differentiation, cells were able to adhere and proliferate with a doubling time of about 6h. The new flagellum emerged from the flagellar pocket around 4h after initiation of cell cycle. Close contact between the flagellar membrane and the flagellar pocket membrane was evident in the intermediate forms. At a later stage of differentiation, intermediate cells exhibited a longer flagellum (shorter than in promastigotes) that contained a PFR and electron dense aggregates in the flagellar matrix. In some cells, PFR profiles were observed inside the flagellar pocket. Taken together, these data contribute to the understanding of flagellum biogenesis and organisation during L. amazonensis differentiation.

Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis.

BACKGROUND: The transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation. METHODS: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression. RESULTS: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. In intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region. CONCLUSIONS: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography.

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

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.