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The Reservosome of Trypanosoma cruzi Epimastigotes: An Organelle of the Endocytic Pathway with a Role on Metacyclogenesis

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Maurilio José Soares
Maurilio José Soares
Maurilio José Soares
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139139
139139
139Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I: 139-141, 1999
The Reservosome of Trypanosoma cruzi Epimastigotes: an
Organelle of the Endocytic Pathway with a Role on
Metacyclogenesis
Maurilio José Soares
Laboratório de Biologia Celular de Microrganismos, Departamento de Ultra-estrutura e Biologia Celular,
Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
Key words: reservosome - Trypanosoma cruzi - endocytosis - metacyclogenesis
Reservosomes are large (0.4-0.6 µm in diam-
eter) membrane-bound organelles found at the pos-
terior end of Trypanosoma cruzi epimastigote
forms. They present a protein-rich, electron dense
matrix, where several tiny round electron lucent
inclusions are immersed. Due to their inner struc-
ture, these organelles were formerly designated as
multivesicular bodies (De Souza 1984). However,
ultrastructural demonstration that the inner inclu-
sions were not membrane bound, together with
cytochemical evidentiation that these vesicles were
indeed lipid droplets, revealed the misinterpreta-
tion of the former nomenclature, and the name
reservosome was then proposed (Soares & De
Souza 1988). Reservosomes are morphologically
and biochemically distinct from the recently de-
scribed acidocalcisomes (Scott et al. 1997). As
reservosomes are a site for protein accumulation,
two major questions arise: how proteins arrive
there, and what reservosomes are for?
THE ENDOCYTIC PATHWAY
Endocytosis of nutrients in trypanosomatid pro-
tozoa is restricted to the flagellar pocket membrane
(Webster & Russel 1993, Radek & Hausmann
1994, Overath et al. 1997). However, epimastigotes
of T. cruzi present an additional site for the uptake
of macromolecules: the cytostome, a deep invagi-
nation of the cell plasma membrane close to the
flagellar pocket region. It appears that the
cytostome is physically linked to the flagellum
(Okuda et al. 1977) and represents the main site
for both receptor-mediated-endocytosis and fluid-
phase-pinocytosis in epimastigotes (Soares & De
Souza 1991, Porto-Carreiro et al. 1998). Endocytic
vesicles bud off from the cytostome and the flagel-
lar pocket membranes and then deliver their cargo
to the reservosomes (Soares & De Souza 1991,
Soares et al. 1992). Reservosomes contain tyrosine-
phosphorylated proteins, suggesting that protein
kinases play a role in the internalization process
(Vieira et al. 1996). Incubation of epimastigotes
with ATP (50 mM, for 24 hr) prior to the addition
of horseradish peroxidase (as a marker of the
endocytic pathway) affected the formation of nor-
mal reservosomes (Bogitsh et al. 1997), possibly
acting on the (still unknown) translocation system
governing the traffic of the small endocytic vesicles
that cargo proteins from the cell surface to the stor-
age organelles.
Reservosomes are acidic organelles, contain-
ing cruzipain (a cysteine proteinase) and ingested
proteins. On this basis, it has been proposed that
these structures are pre-lysosomal compartments
(Soares et al. 1992). Imunocytochemical quantifi-
cation using DAMP as a probe showed that
reservosomes have a luminal pH of about 6.0
(Soares et al. 1992). However, it is still a matter of
speculation how these organelles are acidified, as
they were not labeled with antibodies against a
vacuolar-type H+-ATPase, which however recog-
nized other intracellular vacuoles, possibly the
acidocalcisomes (Benchimol et al. 1998). A 52-
kDa protein sharing sequence homology with glu-
tathione S-transferase (Tc52) has been also local-
ized in reservosomes (Ouassi et al. 1995). It has
been postulated that Tc52 is released from the para-
site to the external milieu, in order to scavenge glu-
tathione (GSH). The Tc52-GSH complex could be
then internalized (by receptor mediated endocyto-
sis?) and accumulated in the reservosomes. As
GSH may serve as a storage and transport form of
cysteine moieties, it was suggested that the Tc52-
GSH complexes might act as a cysteine delivery
system. Accordingly, TC52 is developmentally
regulated, being fully expressed only by the
epimastigotes.
The presence of an early endosomal compart-
ment in T. cruzi epimastigotes is still controver-
sial. It is well known that incubation of the para-
Fax: +55-21-260.4434. E-mail: maurilio@ioc.fiocruz.br
Received 9 June 1999
Accepted 9 August 1999
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140 The Reservosome of T. cruzi Epimastigotes  Maurilio José Soares
sites at 28°C with gold labeled proteins results in
labeling inside cytoplasmic vesicles and tubules,
as well as in the reservosomes (Soares & De Souza
1991, Soares et al. 1992). Figueiredo and Soares
(1996) showed that incubation of the parasites at
12°C (a condition that hinders the fusion of
endocytic vesicles with early endosomes in mam-
malian cells) blocked the pinching of endocytic
vesicles at the cytostome, inhibiting the uptake of
nutrients by the cells. Labeling could be found in
the cytostome, but not inside the flagellar pocket
or intracytoplasmic vesicles. When the tempera-
ture was raised to 28°C, labeling could be then
again found in the reservosomes. From these ex-
periments, it was concluded that early endosomes
are lacking; cargo vesicles coming from the cell
surface (cytostome and flagellar pocket mem-
branes) should shuttle their content directly to the
reservosomes. On the other hand, three-dimen-
sional reconstruction of cytoplasmic tubules and
vesicles located close to the flagellar pocket
showed that they are interconnected, forming a
branched network at the anterior end of the cell,
morphologically similar to the typical mammalian
early endosomes (Porto-Carreiro et al. 1998).
Bogitsh et al. (1996) presented some data dem-
onstrating that, although containing cysteine pro-
teinase, reservosomes are unlikely to be lysosomes
(albeit lysosomes have not yet been clearly mor-
phologically and biochemically defined in
trypanosomatids). The authors showed that incu-
bation of epimastigotes with ammonium chloride
(a weak base that accumulates in acidic compart-
ments) resulted in swelling of reservosomes and
electron-lucent vacuoles (considered as lysos-
omes). However, the exposure period required for
swelling of reservosomes was significantly greater
than that required for the same effect in lysosomes,
probably due to the different pH inside these com-
partments. Furthermore, methyl esters of
aminoacids (which accumulate in eukaryotic ly-
sosomes) had little effect upon reservosomes, pre-
cluding their being lysosomes and suggesting that
the proteolytic enzymes, such as cysteine protein-
ases, can be in an inactive state during a life period
of the parasites.
THE ROLE OF RESERVOSOMES IN METACYCLO-
GENESIS
A stereological study showed that reservosomes
occupy about 6% of the total cell volume of
epimastigotes, but gradually vanish during the dif-
ferentiation process to the trypomastigote form
(Soares et al. 1989, Figueiredo et al. 1994). It has
been suggested that the nutrients accumulated in
the reservosomes could be used as a main energy
source for this activity. A fascinating hypothesis is
that a nutritional stress triggers the acidification of
the luminal content and the activation of the en-
zymes contained inside the reservosomes, which
then evolve to a lysosomal state. Degradation of
stored proteins would then lead to the disappear-
ance of the reservosomes, with the release of amino
acids to the cell cytoplasm. Accordingly, biochemi-
cal data demonstrate that consumption of amino
acids is favored in epimastigotes under starvation
conditions (Urbina 1994).
Reservosomes contain cruzipain (also known
as cruzain and GP57/51), the major cysteine pro-
teinase of T. cruzi. Expression of cruzipain is de-
velopmentally regulated, the enzyme levels being
about 10-fold higher in epimastigotes (Cazzulo et
al. 1997). High expression of reservosomes and
cysteine proteinases in epimastigotes, but not in
trypomastigotes, suggests the participation of
reservosomes in T. cruzi metacyclogenesis. Some
data support this hypothesis: Franke de Cazzulo et
al. (1994) demonstrated that proteinase inhibitors
reduced growth and differentiation of T. cruzi. Ul-
trastructural data showed that treatment of the para-
sites with cysteine proteinase inhibitors arrested
the transport of the enzymes to the reservosomes
at the Golgi complex cisternae level, leading to cell
death (Engel et al. 1998). Preliminary observations
reported by Figueiredo et al. (1998) in epi-
mastigotes maintained in TAU3AAG medium (a
condition that induces metacyclogenesis) showed
that a close relationship exists between uptake of
nutrients, adhesion to the substrate and cell differ-
entiation in T. cruzi.
CONCLUSION
Although the reservosome seems to play a piv-
otal role in the life cycle of T. cruzi, little is still
known about this fascinating organelle. A still
blurry image is slowly coming to sight relative to
the endocytic process of T. cruzi cells, but unfor-
tunately most data has been obtained from
epimastigote forms maintained in culture media.
A more precise characterization of reservosomes
and cytoplasmic vesicles is at the moment diffi-
cult, since specific markers to the endocytic com-
partments are still lacking. Some hope comes from
the recent obtention of a purified subcellular frac-
tion containing reservosomes of T. cruzi
epimastigotes (Cunha-e-Silva et al. 1998). Involve-
ment of this organelle in vital metabolic pathways
of the parasites indicates that reservosomes are
potential targets for the development of chemo-
therapeutic drugs.
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... Cruzipain, a cathepsin L-like cysteine protease detected in all stages of the parasite cell cycle [46], localises within the reservosomes and presents several functions on the homeostasis of the parasite, such as reservosome consumption, thus positively affecting MTG (Figure 2A) [47][48][49]. Autophagy-derived vesicles are necessary for cruzipain trafficking from the ER and the Golgi apparatus into the reservosomes. Fusion among these organelles augments the acidification of the luminal content of reservosomes and MTG depends on a poor environment, stress-related signalling, and autophagy. ...
... Cruzipain, a cathepsin L-like cysteine protease detected in all stages of the parasite cell cycle [46], localises within the reservosomes and presents several functions on the homeostasis of the parasite, such as reservosome consumption, thus positively affecting MTG (Figure 2A) [47][48][49]. Autophagy-derived vesicles are necessary for cruzipain trafficking from the ER and the Golgi apparatus into the reservosomes. Fusion among these organelles augments the acidification of the luminal content of reservosomes and activates hydrolases to cleave stored lipids and proteins such as cruzipain by self-proteolysis. ...
... Increasing levels of dimethylation of H3K76 were also observed during MTG, indicating that this PTM is relevant to the cell cycle [63]. [40], TcIP3R [40,64], Cruzipain [47][48][49], Metacaspase 3 [52], TcSTI1 [53,54], HDAC [60,61], TcSIR2RP1 [62], TcHMGB [65], TcRPA [66,67], Calpain [68,69], TcOGNT2 [70], Tc-eIF2α [71,72], TcZFP2 [73], TcZC3H31 [74] and TcUBP1 [75]. ...
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Chagas disease is a neglected infectious disease caused by the protozoan Trypanosoma cruzi, primarily transmitted by triatomine vectors, and it threatens approximately seventy-five million people worldwide. This parasite undergoes a complex life cycle, transitioning between hosts and shifting from extracellular to intracellular stages. To ensure its survival in these diverse environments, T. cruzi undergoes extreme morphological and molecular changes. The metacyclic trypomastigote (MT) form, which arises from the metacyclogenesis (MTG) process in the triatomine hindgut, serves as a crucial link between the insect and human hosts and can be considered the starting point of Chagas disease. This review provides an overview of the current knowledge regarding the parasite’s life cycle, molecular pathways, and mechanisms involved in metabolic and morphological adaptations during MTG, enabling the MT to evade the immune system and successfully infect human cells.
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... Reservosomes are organelles similar lysosomes. Their morphology varies according to the parasite strain but are generally round (Soares 1999). They are membrane-bound and consist of an electron-dense matrix with vesicles and lipid inclusions and acid hydrolases such as cruzipain (Soares 1999). ...
... Their morphology varies according to the parasite strain but are generally round (Soares 1999). They are membrane-bound and consist of an electron-dense matrix with vesicles and lipid inclusions and acid hydrolases such as cruzipain (Soares 1999). Reservosomes store secretory proteins and macromolecules and endocytosed nutrients and, therefore, it is a nutrient reserve site (Cunha-e-Silva et al.2006). ...
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Luminescent semiconductor nanocrystals or quantum dots (QDs) emerge as important fluorescent probes for in vitro and in vivo Trypanosoma cruzi cells studies. However, to ensure applicability to living organisms, several tests still need to be done. Since several toxic events are caused by QDs, such as loss of mitochondrial membrane potential, ROS generation, DNA damage and cell death by autophagy. We performed a review of the literature on mechanisms of cellular uptake, internalization and citotoxicity of nanoparticles including our results about the evaluation of biological toxicity in T. cruzi. We evaluated the possible effects on parasite growth curves in a time - scale of control and incubated cells with different concentrations of CdTe – QDs (0.2; 2.0; 20 and 200µM) to determine the development cells changes. In addition, intracellular ROS were measured by Electron Paramagnetic Resonance Spectroscopy (EPR) technique. According our results, we can infer that the toxic effects of QDs in T. cruzi are dose-dependent and that high levels of ROS are involved in cellular toxicity promoted by higher concentrations of QDs. In summary, parasites labeled with low concentrations of nanoparticles are suitable and can be used as bioimaging tools for living parasites. However, more studies on QDs cytotoxicity need to be carried out.
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... The translucent vacuoles observed by light microscopy at the posterior end of epimastigotes corresponded to the large, electron-lucent vacuoles found by transmission electron microscopy. Incubation with acridine orange showed an approximately 99% increase of the fluorescence signal by flow cytometry and a stronger red labeling by fluorescence microscopy at the posterior cell end, indicating that these large vacuoles correspond to the reservosomes, acidic organelles that accumulate ingested proteins [55,56]. The lower endocytic capacity of 2-BP-treated epimastigotes resulted in the appearance of these less dense reservosomes. ...
... It has been proposed that the content of reservosomes is metabolized during the metacyclogenesis process [56]. Considering that the 2-BP-treated epimastigotes had poor endocytic activity, we analyzed the effect of incubating epimastigotes with 2-BP before metacyclogenesis (pre-stress assay). ...
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... They also contain several hydrolases that are acquired from the secretory pathway and crucial for the digestion of the endocytosed material [4,5]. Reservosomes disappear during the differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) [6,7], a process that can be triggered by nutritional stress [8]. It is possible that the content of reservosomes is mobilized as energy source during metacyclogenesis. ...
... Nutritional stress would trigger reservosome acidification, activating its hydrolases and altering organelle activity from a 'pre' to a 'full' lysosomal state. The consumption of stored material triggered by nutritional stress would explain reservosome disappearance during metacyclogenesis [7]. However, a recent study suggested that LROs from metacyclic trypomastigotes may arise directly from reservosomes [9]. ...
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... Proteomic analysis showed that the proteomic profile between parasites differentiated in TAU 3AAG and TAU Pro medium is very similar, which could be expected since Pro is During nutritional stress and metacyclogenesis, translation is attenuated [57], T. cruzi uses proteins previously accumulated in reservosomes as amino acid sources [58] and, accordingly, inhibition of proteasome activity prevents metacyclogenesis [59]. ...
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... The glucose depletion in medium leads to the elongation of Epi flagella (Tyler and Engman 2000), which may be a prestage to adhesion or an adaptation that allows greater capture of nutrients from the medium by increasing the membrane area (Hernández et al. 2012). In addition, the reservosome, a structure where lipids and proteins are stored, decreases in size, indicating that this stored content is consumed during differentiation (Soares 1999). A more recent study has shown ultrastructural changes in parasites subjected to NS, with a slimmer appearance, alterations in the location and structure of mitochondrion-kinetoplast, and the accumulation of vesicles in the cytoplasm of the parasites (Pérez-Morales et al. 2017). ...
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... cruzi entre ellos) poseen también una organela llamada glicosoma (similar al peroxisoma), donde ocurre la mayor parte de la vía glucolítica, y otra organela denominada acidocalcisoma, que cumple la función de reservorio de calcio, fosfatos y otras sales inorgánicas [18]. Una tercera organela distintiva es el reservosoma, compuesta por vesículas estrechamente relacionadas al proceso de endocitosis, en donde se acumulan proteínas que sólo se encuentran en el estadio epimastigote [74]. Por debajo de la membrana plasmática, se encuentra una capa de microtúbulos sub-peliculares, probablemente responsables de la rigidez celular [15]. ...
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... Trypanosoma cruzi, the aetiological agent of Chagas disease, presents special features when compared with other eukaryotes, like a single mitochondrion that presents interlocked DNA circles (kDNA) contained in the kinetoplast and a typical cytoskeleton array (Soares, 1999;De Souza, 2002). The arrangement of trypanosomatid cytoskeleton is simpler than those of most other eukaryotic cells; however, it is precisely organized and constituted by stable microtubules. ...
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Full-text available
  • Nov 2018
Trypanosoma cruzi , the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.
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Imidazoles and Oxazoles from Lapachones and Phenanthrene‐9,10‐dione: A Journey through their Synthesis, Biological Studies, and Optical Applications
Article
  • Jun 2021
Diverse structural frameworks are found in natural compounds and are well known for their chemical and biological properties; such compounds include the imidazoles and oxazoles. Researchers worldwide are continually working on the development of methods for synthesizing new molecules bearing these basic moiety and evaluating their properties and applications. To expand the knowledge related to azoles, this review summarizes important examples of imidazole and oxazole derivatives from 1,2‐dicarbonyl compounds, such as lapachones and phenanthrene‐9,10‐diones, not only regarding their synthesis and biological applications but also their photophysical properties and uses. The data concerning the latter are particularly scarce in the literature, which leads to underestimation of the potential applications that can be envisaged for these compounds. Strategies and reactions for preparing imidazole and oxazole derivatives from lapachones and phenanthrene‐9,10‐dione are described. These compounds were used as a template to study their biological applications, photophysical properties, and bioimaging studies. In this review, we also discuss the wide range of applications of these important heterocycles.
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Conservation and divergence within the clathrin interactome of Trypanosoma cruzi
Article
Full-text available
  • Aug 2016
Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent.
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The reservosomes of epimastigote forms ofTrypanosoma cruzi: occurrence during in vitro cultivation
Article
Full-text available
  • Jun 1994
Reservosomes are large membrane-bound structures found mainly at the posterior end of epimastigote forms ofTrypanosoma cruzi, the agent of Chagas'' disease. We screened 5-day-old culture forms of the following strains at the ultrastructural level for the presence of reservosomes:T. cruzi strains Y and YuYu as well asTrypanosoma (Schizotrypanum) spp. strains M431, M504, and M519 isolated from bats (Epitesicus sp.) in Santa Catarina, Brazil. The organelles could be found in all strains analyzed, supporting a previous hypothesis that they are a marker of trypanosomes of theSchizotrypanum subgenus. Stereological analysis was carried out with the Y strain to follow the development of reservosomes and lipid droplets during in vitro cultivation of the parasites. Reservosomes were partitioned to the daughter cells during cell division such that the organelles were present in newly formed parasites. Estimation of the volume density after 3, 5, 7, 9, and 12 days of cultivation showed that it was lower in younger cultures, becoming maximal at day 9 (8.0%), but decreased in older cultures (5.9% at day 12). Morphological changes also occurred: type I reservosomes presented an electron-dense matrix with lipid droplets and were characteristic of younger cultures, whereas type II reservosomes presented a homogeneous matrix without lipid inclusions and predominated in older cultures. These organelles were absent in bloodstream trypomastigote forms isolated from infected mice. Lipid inclusions were found in larger numbers at the 3rd and 12th days of culture, but their volume density was lower at the log phase of growth.
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Identification of a large-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi
Article
Full-text available
  • Jun 1992
Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4 degrees C or 28 degrees C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytosome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and lgp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.
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Trypanosoma cruzi: A 52-kDa Protein Sharing Sequence Homology with Glutathione S-Transferase Is Localized in Parasite Organelles Morphologically Resembling Reservosomes
Article
Full-text available
  • Jan 1996
We have previously isolated and characterized a Trypanosoma cruzi cDNA encoding a polypeptide with a molecular mass of 52 kDa (Tc52) sharing significant homology to glutathione S-transferase. In the present study, by molecular and immunological approaches, we showed that Tc52 is preferentially expressed by dividing forms of the parasite: (e.g., epimatigotes and amastigotes). Moreover, we could identify the reactive antigen in different T. cruzi strains. A different pattern of reactivity on immunoblots was observed in the case of Trypanosoma rangeli. Furthermore, immunofluorescence assays using T. cruzi epimastigote culture forms revealed that the reactive antigen is localized within cytoplasmic organelles morphologically ressembling the structures previously designated as the reservosome found mostly at the posterior end of the parasite. Furthermore, the antibodies did not react against trypomastigotes which emerged from infected fibroblasts, whereas amastigotes showed polar fluorescence. Immunogold labeling and electron micrographs further revealed that the Tc52 protein is mainly associated with organelles composed of a large network of multivesicular structures, the latter being more abundant in epimastigotes. Taken together, these results demonstrated that Tc52 is associated with organelles composed of a multivesicular network and appears to be developmentally regulated, being fully expressed by parasite dividing forms.
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Endocytosis and secretion in trypanosomatid parasites — Tumultuous traffic in a pocket
Article
  • Feb 1997
  • TRENDS CELL BIOL
Trypanosomatids are flagellated protozoan parasites of invertebrates, vertebrates and plants. Some species, found in the subtropics and tropics, cause chronic diseases in humans and domestic animals. The surface of the trypanosomatid provides a shield against environmental challenges, ligands for interaction with host cells, as well as receptors and transporters for the uptake of nutrients. Communication between the parasite and its environment is confined to the flagellar pocket, an invagination of the plasma membrane around the base of the flagellum. In this review, the authors discuss endocytosis, secretion and membrane trafficking in Trypanosoma and Leishmania.
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Endocytosis of gold-labeled proteins and LDL by
Article
  • Feb 1991
Endocytosis was studied at the ultrastructural level in different developmental forms of Trypanosoma cruzi after incubation of the parasites in the presence of gold-labeled proteins (albumin-Au, peroxidase-Au and transferrin-Au) and low-density lipoprotein (LDL-Au). Epimastigote (culture) forms actively ingested LDL and proteins. Initially, gold particles were seen adhering only to the cytostome and inside the flagellar pocket. In parasites incubated at 4 degrees C with transferrin-Au or peroxidase-Au, labeling was found only at these two sites, showing that receptor-mediated endocytosis occurs in both regions. In the cytoplasm, gold particles were seen only inside two different compartments: membrane-bound vesicles and reservosomes. Incubation of epimastigotes with acridine orange followed by fluorescence microscopy revealed intense orange staining, indicating that the reservosomes have an acidic pH. This staining was abolished after incubation of the parasites in the presence of ammonium chloride. These data confirm that this compartment is the site of accumulation of ingested lipids and proteins. Little intracellular labeling with transferrin-Au was found in in vitro-derived amastigotes and trypomastigotes (both lack reservosomes). However, although in amastigotes very few gold particles were seen bound to the cells, in trypomastigotes they were observed bound to the membrane that encloses the cell body, the flagellar pocket, and the flagellum, suggesting that the receptors are more abundant in this form.
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A stereological study of the differentiation process in Trypanosoma cruzi
Article
  • Feb 1989
When epimastigote forms of Trypanosoma cruzi grown in a rich medium (LIT) are transferred to a simple, chemically defined medium (TAU3AAG, containing Ca2+, Mg2+, K+, Na+, L-proline, L-glutamate, and L-aspartate in phosphate buffer) they transform into trypomastigote forms. Morphometric analysis of transmission electron micrographs of thin sections of parasites collected at different steps of the transformation process showed that no changes occurred in the volume density of mitochondria and cytoplasmic vacuoles. However, a significant increase in the volume density of the kinetoplast DNA network as well as the lipid inclusions and a decrease in that of the reservosome (a special type of endosome) was observed. These observations suggest that during differentiation, T. cruzi accumulates lipids and uses molecules contained in the reservosome as its main energy source.
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Cytoplasmic organelles of Trypanosomatids: a cytochemical and stereological study
Article
  • May 1988
  • J Submicr Cytol Pathol
The cytoplasmic organelles of different protozoa of the family Trypanosomatidae were characterized by ultrastructural cytochemistry and stereology. Data were obtained for mitochondria, lipid inclusions, glycosomes (peroxisome-like organelle), empty membrane-bounded vacuoles, reservosomes of Trypanosoma spp., multivesicular body of Crithidia fasciculata and dense granules of Crithidia oncopelti. The stereological analysis (D = mean diameter, Vv = volume density and Nv = numerical density) was performed in glutaraldehyde-formaldehyde and osmium tetroxide-potassium ferricyanide fixed parasites, which showed an excellent preservation of the membranes and cytoplasmic organelles. Lipid inclusions, not limited by a unit membrane, appeared electron-dense after post-fixation in an osmium-imidazole buffered solution. Catalase, a peroxisomal enzyme, was detected only in the glycosomes of the lower trypanosomatids. Empty membrane-bounded vacuoles showed positive reaction when the cells were incubated in a medium specific for the detection of the lysosomal enzyme acid phosphatase. The reservosomes of Trypanosoma spp., sub-genus Schizotrypanum, could be differentiated from the multivesicular bodies of other trypanosomatids, since they lack true vesicles. They contain lipid inclusions dispersed in an electron-dense matrix which stained positively when the cells were incubated in ethanolic phosphotungstic acid to detect basic proteins.
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Novel Cell Biology of Trypanosoma cruzi
Article
  • Feb 1984
  • INT REV CYTOL
This paper reviews basic aspects of the cell biology of trypanosomatids with a special emphasis on results obtained with Trypanosoma cruzi. The following structures are discussed: (a) the various domains of the cell surface that can be identified using freeze-fracture and cytochemistry, and the association of the plasma membrane with microtubules; (b) the organization of the flagellum, especially the paraflagellar rod; (c) the structure of the nucleus and its behaviour during cell division; (d) the kinetoplast and the replication of the kinetoplast DNA; (e) the organization of the glycosome and the import of its proteins; (f) the endocytic pathway, emphasizing the role played by the cytostome and the reservosomes; (g) the organization, distribution and function of the acidocalcisomes.
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Effect of proteinase inhibitors on growth and differentiation of Trypanosoma cruzi
Article
  • Dec 1994
Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.
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