Narcisa L. Cunha-e-Silva

Trypanosoma cruzi epimastigote endocytic pathway: cargo enters the cytostome and passes through an early endosomal network before storage in reservosomes.

It has been known for many years that trypanosomatids require exogenous essential growth factors in order to divide. Two surface domains are involved in starting nutrient endocytosis: the flagellar pocket and the cytostome. Although the flagellar pocket plays a fundamental role in the endocytic process occurring in several trypanosomatids, we have shown the cytostome as the main structure involved in this process in epimastigote forms of T. cruzi. After one minute of endocytosis, cargo is still found at the cytostome entry as well as along the cytopharynx. After two, five and fifteen minutes of endocytosis, cargo was seen inside vesicles and tubules, prior to fusing with reservosomes. Three-dimensional reconstruction of these tubules and vesicles showed they are interconnected, forming an intricate and branched network, distributed from the perinuclear region to the posterior end of the cell. Whole unfixed parasites that had taken up gold-protein conjugates for fifteen minutes were washed and dried on electron microscope grids. Observation with an energy-filtering transmission electron microscope revealed long gold-filled tubules at the posterior end of the cell. Parasites treated with ammonium chloride had their intracellular traffic slowed down, which allowed us to observe many events of vesicle fusion. The acidic nature of this network was evidenced using acridine orange. Based on pH and protein uptake kinetics we propose that the vesicular-tubular network is the early endosome of Trypanosoma cruzi epimastigotes.

Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells

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 β-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 (∼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.

Subcellular proteomics of Trypanosoma cruzi reservosomes

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.

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

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.

All Trypanosoma cruzi developmental forms present lysosome-related organelles

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa.

ABSTRACT Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.

Reservosomes: multipurpose organelles?

Reservosomes are endocytic organelles from Trypanosoma cruzi epimastigotes that store proteins and lipids for future use. The lack of molecular markers for the compartments of this parasite makes it difficult to clarify all reservosome functions, as they present characteristics of pre-lysosomes, lysosomes and recycling compartments.

Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM)

The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen’s topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.

Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope-tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.