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.

Alterations on growth and cell organization of Giardia intestinalis trophozoites after treatment with KH-TFMDI, a novel class III histone deacetylase inhibitor.

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis.

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.

Structures containing galectin-3 are recruited to the parasitophorous vacuole containing Trypanosoma cruzi in mouse peritoneal macrophages.

Trypanosoma cruzi has a complex life cycle where the infective forms for the vertebrate host are trypomastigotes and amastigotes. Both forms invade and lyse their parasitophorous vacuole (PV) membrane, entering into the cytoplasm of its host cells. Galectin-3 (Gal-3) is a protein abundantly distributed in macrophages and epithelial cells. Previous studies demonstrated that Gal-3 binds to a 45KDa mucin of trypomastigotes surface, enhancing its adhesion to the extracellular matrix and even its entry into cells. Gal-3 has another novel cytoplasmic function recently described: a vacuole lyses marker in intracellular bacteria. Considering (1) the importance of Gal-3 during T. cruzi early infection and (2) the importance of T. cruzi PV lyses for parasite differentiation and replication, this study intended to explore a possible recruitment of structures containing Gal-3 (G3CSs) to T. cruzi PVs. Microscopy analyses showed these G3CSs around PVs after 30 and 90 min of amastigotes and trypomastigotes infection, respectively. This recruitment was specific for T. cruzi PVs since we did not observe the same distribution at macrophages vacuoles containing fluorescent microspheres (FM). Concomitantly, this study intended to analyze the participation of actin cytoskeleton in T. cruzi PV maturation. We observed that actin filaments form a "belt-like" structure around trypomastigotes and amastigotes PVs, also labeled for Gal-3. At the time proposed for PV lysis, we observed an actin disassembling while LAMP-1 was recruited to PVs membrane. However, this pattern was maintained in macrophages derived from Gal-3 knockout mice, revealing that the actin belt structure forms independently from Gal-3. Taken together, these data suggest that G3CSs are recruited to vicinity of T. cruzi PV and that actin filaments localize and remain around T. cruzi PVs until the time of its lysis.

Inhibition of NAD+-dependent histone deacetylases (sirtuins) causes growth arrest and activates both apoptosis and autophagy in the pathogenic protozoan Trypanosoma cruzi.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, affects approximately 7-8 million people in Latin America. The drugs available to treat this disease are ineffective against chronic phase disease and are associated with toxic side effects. Therefore, the development of new compounds that can kill T. cruzi at low concentrations is critically important. Herein, we report the effects of a novel 3-arylideneindolin-2-one that inhibits sirtuins, which are highly conserved proteins that are involved in a variety of physiological processes. The compound KH-TFMDI was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi, and its effects were evaluated using flow cytometry, light and electron microscopy. KH-TFMDI inhibited the replication of T. cruzi intracellular amastigotes with an IC50 of 0.5 ± 0.2 µM, which is significantly lower than the IC50 of benznidazole. The compound also lysed the highly infectious bloodstream trypomastigotes (BST) with LC50 values of 0.8 ± 0.3 µM at 4 °C and 2.5 ± 1.1 µM at 37 °C. KH-TFMDI inhibited cytokinesis and induced several morphological changes in the parasite, leading to its death by apoptosis and autophagy. This study highlights sirtuins as a potential new target for Chagas disease therapy.

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasite contains a single nucleus and a kinetoplast, which harbors the mitochondrial DNA (kDNA). DNA topoisomerases act during replication, transcription and repair and modulate DNA topology by reverting supercoiling in the DNA double-strand. In this work, we evaluated the effects promoted by camptothecin, a topoisomerase I inhibitor that promotes protozoan proliferation impairment, cell cycle arrest, ultrastructure alterations and DNA lesions in epimastigotes of T. cruzi. The results showed that inhibition of cell proliferation was reversible only at the lowest drug concentration (1µM) used. The unpacking of nuclear heterochromatin and mitochondrion swelling were the main ultrastructural modifications observed. Inhibition of parasite proliferation also led to cell cycle arrest, which was most likely caused by nuclear DNA lesions. Following camptothecin treatment, some of the cells restored their DNA, whereas others entered early apoptosis but did not progress to late apoptosis, indicating that the protozoa stay alive in a "senescence-like" state. This programmed cell death may be associated with a decrease in mitochondrial membrane potential and an increase in the production of reactive oxygen species. Taken together, these results indicate that the inhibition of T. cruzi proliferation is related to events capable of affecting cell cycle, DNA organization and mitochondrial activity.