Differentiating Trypanosoma cruzi in a Host Mammalian Cell Imaged in Aqueous Liquid by Atmospheric Scanning Electron Microscopy.

Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites. IMPORTANCE Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.

Drug repurposing for Chagas disease: In vitro assessment of nimesulide against Trypanosoma cruzi and insights on its mechanisms of action.

Chagas disease is a neglected illness caused by Trypanosoma cruzi and its treatment is done only with two drugs, nifurtimox and benznidazole. However, both drugs are ineffective in the chronic phase, in addition to causing serious side effects. This context of therapeutic limitation justifies the continuous research for alternative drugs. Here, we study the in vitro trypanocidal effects of the non-steroidal anti-inflammatory drug nimesulide, a molecule that has in its chemical structure a toxicophoric nitroaromatic group (NO2). The set of results obtained in this work highlights the potential for repurposing nimesulide in the treatment of this disease that affects millions of people around the world.

Furan derivatives impair proliferation and affect ultrastructural organization of Trypanosoma cruzi and Leishmania amazonensis.

Chagas disease and leishmaniasis are neglected diseases caused by parasites of the Trypanosomatidae family and together they affect millions of people in the five continents. The treatment of Chagas disease is based on benznidazole, whereas for leishmaniasis few drugs are available, such as amphotericin B and miltefosine. In both cases, the current treatment is not entirely efficient due to toxicity or side effects. Encouraged by the need to discover valid targets and new treatment options, we evaluated 8 furan compounds against Trypanosoma cruzi and Leishmania amazonensis, considering their effects against proliferation, infection, and ultrastructure. Many of them were able to impair T. cruzi and L. amazonensis proliferation, as well as cause ultrastructural alterations, such as Golgi apparatus disorganization, autophagosome formation, and mitochondrial swelling. Taken together, the results obtained so far make these compounds eligible for further steps of chemotherapy study.

Synergistic effect and ultrastructural changes in Trypanosoma cruzi caused by isoobtusilactone A in short exposure of time.

Butanolides have shown a variety of biological effects including anti-inflammatory, antibacterial, and antiprotozoal effects against certain strains of Trypanosoma cruzi. Considering the lack of an effective drug to treat T. cruzi infections and the prominent results obtained in literature with this class of lactones, we investigated the anti-T. cruzi activity of five butanolides isolated from two species of Lauraceae, Aiouea trinervis and Mezilaurus crassiramea. Initially, the activity of these compounds was evaluated on epimastigote forms of the parasite, after a treatment period of 4 h, followed by testing on amastigotes, trypomastigotes, and mammalian cells. Next, the synergistic effect of active butanolides against amastigotes was evaluated. Further, metacyclogenesis inhibition and infectivity assays were performed for the most active compound, followed by ultrastructural analysis of the treated amastigotes and trypomastigotes. Among the five butanolides studied, majoranolide and isoobtusilactone A were active against all forms of the parasite, with good selectivity indexes in Vero cells. Both butanolides were more active than the control drug against trypomastigote and epimastigote forms and also had a synergic effect on amastigotes. The most active compound, isoobtusilactone A, which showed activity against all tested strains inhibited metacyclogenesis and infection of new host cells. In addition, ultrastructural analysis revealed that this butanolide caused extensive damage to the mitochondria of both amastigotes and trypomastigotes, resulting in severe morphological changes in the infective forms of the parasite. Altogether, our results highlight the potential of butanolides against the etiologic agent of Chagas disease and the relevance of isoobtusilactone A as a strong anti-T. cruzi drug, affecting different events of the life cycle and all evolutionary forms of parasite after a short period of exposure.

Novel N,N-di-alkylnaphthoimidazolium derivative of ß-lapachone impaired Trypanosoma cruzi mitochondrial electron transport system.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benznidazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel ß-lapachone-derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC50/24 h in the range of 0.8-7.9 µM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled H2O2 release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydrogenase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.

Proteomic changes in Trypanosoma cruzi epimastigotes treated with the proapoptotic compound PAC-1.

Apoptosis is a highly regulated process of cell death in metazoans. Therefore, understanding the biochemical changes associated with apoptosis-like death in Trypanosoma cruzi is key to drug development. PAC-1 was recently shown to induce apoptosis in T. cruzi; with this as motivation, we used quantitative proteomics to unveil alterations of PAC-1-treated versus untreated epimastigotes. The PAC-1 treatment reduced the abundance of putative vesicle-associated membrane protein, putative eukaryotic translation initiation factor 1 eIF1, coatomer subunit beta, putative amastin, and a putative cytoskeleton-associated protein. Apoptosis-like signaling also increases the abundance of proteins associated with actin cytoskeleton remodeling, cell polarization, apoptotic signaling, phosphorylation, methylation, ergosterol biosynthesis, vacuolar proteins associated with autophagy, and flagellum motility. We shortlist seventeen protein targets for possible use in chemotherapy for Chagas disease. Almost all differentially abundant proteins belong to a family of proteins previously associated with apoptosis in metazoans, suggesting that the apoptotic pathway's key functions have been preserved from trypanosomatids and metazoans. SIGNIFICANCE: Approximately 8 million people worldwide are infected with Trypanosoma cruzi. The treatment of Chagas disease comprises drugs with severe side effects, thus limiting their application. Thus, developing new pharmaceutical solutions is relevant, and several molecules targeting apoptosis are therapeutically efficient for parasitic, cardiac, and neurological diseases. Apoptotic processes lead to specific morphological features that have been previously observed in T. cruzi. Here, we investigate changes in epimastigotes' proteomic profile treated with the proapoptotic compound PAC-1, providing data concerning the regulation of both metabolic and cellular processes in nonmetazoan apoptotic cells. We shortlist seventeen protein target candidates for use in chemotherapy for Chagas disease.

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.

Antichagasic effect of hemocyanin derived from antimicrobial peptides of penaeus monodon shrimp.

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Crystal structure of Q4D6Q6, a conserved kinetoplastid-specific protein from Trypanosoma cruzi.

Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.

Lippia sidoides and Lippia origanoides essential oils affect the viability, motility and ultrastructure of Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered a public health problem. The current chemotherapy for this illness causes serious side effects and its use in the chronic phase of the disease is still controversial. In this regard, the investigation of novel therapeutic strategies remains a priority. The essential oils (EOs) from aromatic plants emerge as a promising source of bioactive compounds. In a previous work we reported the trypanocidal activity of the essential oils from the medicinal plants Lippia sidoides (LSEO) and Lippia origanoides (LOEO) against T. cruzi. Herein, we aimed to further investigate, in more details, the mode of action of LSEO and LOEO on the different developmental stages of this parasite. We showed that Lippia sidoides (LSEO) and Lippia origanoides (LOEO) induced a significant reduction in the percentage of macrophages infected by T. cruzi and in the number of intracellular parasites. Ultrastructural analysis showed that the treatment with both oils caused morphological changes consistent with loss of viability and cell death. The reduced staining with calcein and the increase in the proportion of HE-positive cells also demonstrated that LSEO and LOEO caused loss of parasite viability and membrane integrity. A considerable decrease in Rhodamine 123 and an increase in fluorescence intensity of MitoSox in LOEO were indicative of loss of mitochondrial potential and generation of reactive oxygen species, which ultimately lead to parasite death. Moreover, the optical tweezer analysis indicated that LOEO was more effective in reducing the motility of the epimastigotes. Taken together, our results demonstrated that the LSEO and LOEO are active against T. cruzi and constitute a promising drugs for the therapy of Chagas disease.

In vitro activities of adamantylidene-substituted alkylphosphocholine TCAN26 against Trypanosoma cruzi: Antiproliferative and ultrastructural effects.

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.

The effects of endoplasmic reticulum stressors, tunicamycin and dithiothreitol on Trypanosoma cruzi.

In higher eukaryotic cells, pertubations in ER environment, called ER stress, usually activate unfolded protein response (UPR) pathway in an attempt to re-stablish the ER homeostasis and prevent cell death. Because trypanosomatids appear to lack the classical UPR, it is not clear how these parasites respond to ER stress. Thus, the aim of this work was to evaluate the effects of ER stressors tunicamycin (TM) or dithiothreitol (DTT) on Trypanosoma cruzi. The TM treatment showed strong trypanostatic effect. At 2.5 µg/mL of TM, the mRNA levels of both binding protein (BiP) and calreticulin (CRT) increased significantly, whereas the protein levels of BiP remained stable. TM treatment induced ultrastructural changes compatible with an autophagic process. The DTT treatment inhibited the cell growth, induced drastic morphological changes, mitochondrial membrane depolarization and increased ROS production. The expression of BiP apparently was not affected by DTT, whereas the mRNA levels of BiP and CRT were significantly reduced. Our results suggest that TM induces autophagy/ER-phagy without causing substantial injury to the parasite. Conversely, the DTT treatment seems to rupture the mitochondrion homeostasis leading to parasite death. The comprehension of the mechanisms behind the susceptibility of T. cruzi to ER stress open perspectives for the development of chemotherapeutic agents addressed to these pathways.

Coumarins isolated from Calophyllum brasiliense produce ultrastructural alterations and affect in vitro infectivity of Trypanosoma cruzi.

BACKGROUND: The current drugs for Chagas Disease caused by the protozoan Trypanosoma cruzi have limited therapeutic potential and are associated with serious side effects. Natural products can aid to develop new chemotherapeutic agents. Several natural coumarins, especially Mammea A/BA, have shown significant activity against T. cruzi and low toxicity on human lymphocytes, but its effectivity on a wide range of strains need to be tested, as well as to deepen in their mode of action and safety. HYPOTHESIS/PURPOSE: To discern the effects and explore the action mechanisms of mammea A/BA and a mixture of mammea coumarins isolated from Calophyllum brasiliense on Mexican strains of T. cruzi belonging to different genotypes and compare its effectivity with the drug benznidazole. STUDY DESIGN: We evaluated the trypanocidal activity in vitro of mammea A/BA (93.6%), and a mixture of coumarins, mammea A/BA + A/BB + A/BD (86:10:1%) on Mexican T. cruzi strains belonging to different genotypes Ninoa, Querétaro (TcI) and Ver6 (TcVI). MATERIAL AND METHODS: Mammea A/BA and the mixture of coumarins, were isolated from Calophyllum brasiliense, identified by proton NMR and purity determined by HPLC. The in vitro trypanocidal activity was evaluated on mobility, growth recovery, morphology and infectivity of T. cruzi. The cytotoxicity on mammalian cells was compared with benznidazole. The ultrastructure of the treated epimastigotes was analyzed by transmission electron microscopy (TEM). RESULTS: Mammea A/BA and the mixture of coumarins showed high trypanocidal activity, affecting the mobility, growth recovery, morphology, ultrastructure of epimastigotes, and drastically reduce trypomastigotes infectivity on Vero cells. These substances were four times more potent than benznidazole and showed low cytotoxicity and high selectivity index. The TEM showed severe alterations on the plasmatic membrane, nuclear envelope, as well as, mitochondrial swelling, that leads to the death of parasites. CONCLUSION: Mammea A/BA (93.6%) and a mixture of mammea A/BA + A/BB and A/BD (86: 10: 1%) isolated from the tropical tree C. brasiliense showed higher trypanocidal activity than the current drug benznidazole on three Mexican strains of T. cruzi. These compounds induced severe physiological and morphological alterations. These results suggest their possible use in preclinical studies.

Morphobiological, morphometric and ultrastructural characterization of sylvatic Trypanosoma cruzi isolates from Rio de Janeiro state, Brazil / Caracterização morfobiológica, morfométrica e ultraestrutural de isolados silvestres de Trypanosoma cruzi do estado do Rio de Janeiro, Brasil

Abstract Triatoma vitticeps is a triatomine with geographic distribution restrict to Brazil, which exhibits high prevalence of Trypanosoma cruzi natural infection. Of special epidemiologic concern, this species often invades households in the states of Rio de Janeiro, Minas Gerais and Espírito Santo. The objective of this study was to evaluate morphological and ultrastructural parameters on three T. cruzi isolates obtained from wild T. vitticeps specimens. The growth and cell differentiation of the parasite was evaluated through epimastigote and trypomastigote forms obtained in the growth curves for three distinct isolates. The maximum growth showed differences at the 20th day of the curve. Our in vitro results show a heterogeneity, regarding these features for samples cultivated under the same conditions. Morphometric analyzes based on the shape of epimastigotes and trypomastigotes corroborated such differentiation. These results highlight the need of better understanding the meaning of this diversity under an eco-epidemiological perspective.

Resumo Triatoma vitticeps é um triatomíneo com distribuição geográfica restrita ao território brasileiro, apresentando alta prevalência de infecção natural pelo Trypanosoma cruzi. Esta espécie é relevante sob o ponto de vista epidemiológico por invadir domicílios com frequência nos estados do Rio de Janeiro, Minas Gerais e Espírito Santo. O objetivo deste estudo foi avaliar parâmetros morfológicos e ultraestruturais, em três isolados de T. cruzi obtidos a partir de triatomíneos silvestres. O crescimento e a diferenciação celular do parasita foi avaliado através das formas epimastigotas e tripomastigotas obtidas nas curvas de crescimento para os três isolados. O crescimento máximo mostrou diferenças no 20º dia da curva. Nossos resultados in vitro mostram uma heterogeneidade, em relação a essas características para amostras cultivadas nas mesmas condições. As análises morfométricas baseadas na conformação de epimastigotas e trypomastigotes corroboraram essa diferenciação. Estes resultados ressaltam a necessidade de uma melhor compreensão do significado desta diversidade sob uma perspectiva eco-epidemiológica.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity.

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi.

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Morphobiological, morphometric and ultrastructural characterization of sylvatic Trypanosoma cruzi isolates from Rio de Janeiro state, Brazil.

Triatoma vitticeps is a triatomine with geographic distribution restrict to Brazil, which exhibits high prevalence of Trypanosoma cruzi natural infection. Of special epidemiologic concern, this species often invades households in the states of Rio de Janeiro, Minas Gerais and Espírito Santo. The objective of this study was to evaluate morphological and ultrastructural parameters on three T. cruzi isolates obtained from wild T. vitticeps specimens. The growth and cell differentiation of the parasite was evaluated through epimastigote and trypomastigote forms obtained in the growth curves for three distinct isolates. The maximum growth showed differences at the 20th day of the curve. Our in vitro results show a heterogeneity, regarding these features for samples cultivated under the same conditions. Morphometric analyzes based on the shape of epimastigotes and trypomastigotes corroborated such differentiation. These results highlight the need of better understanding the meaning of this diversity under an eco-epidemiological perspective.

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.

TrypanocidalActivity of Natural Sesquiterpenoids Involves Mitochondrial Dysfunction, ROS Production and Autophagic Phenotype in Trypanosomacruzi.

Chagas disease is a neglected tropical disease that is caused by the protozoan Trypanosomacruzi and represents a serious health problem, especially in Latin America. The clinical treatment of Chagas disease is based on two nitroderivatives that present severe side effects and important limitations. In folk medicine, natural products, including sesquiterpenoids, have been employed for the treatment of different parasitic diseases. In this study, the trypanocidal activity of compounds isolated from the Chilean plants Drimys winteri, Podanthus mitiquiand Maytenus boaria on three T. cruzi evolutive forms (epimastigote, trypomastigote and amastigote) was evaluated. Total extracts and seven isolated sesquiterpenoids were assayed on trypomastigotes and epimastigotes. Polygodial (Pgd) from D. winteri, total extract from P. mitiqui (PmTE) and the germacrane erioflorin (Efr) from P. mitiqui were the most bioactive substances. Pgd, Efr and PmTE also presented strong effects on intracellular amastigotes and low host toxicity. Many ultrastructural effects of these substances, including reservosome disruption, cytosolic vacuolization, autophagic phenotype and mitochondrial swelling (in the case of Pgd), were observed. Flow cytometric analysis demonstrated a reduction in mitochondrial membrane potential in treated epimastigotes and an increase in ROS production and high plasma membrane permeability after treatment with Pgd. The promising trypanocidal activity of these natural sesquiterpenoids may be a good starting point for the development of alternative treatmentsforChagas disease.