Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Pereira, Miria G.; Nakayasu, Ernesto; Sant’Anna, Celso; Cicco, Nuccia N.T. De; Atella, Geórgia C.; Souza, Wanderley de; Almeida, Igor C.; Cunha-e-Silva, Narcisa L.

doi:10.1371/journal.pone.0022359

Cited by 47 publications

(29 citation statements)

References 56 publications

(75 reference statements)

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“…Epimastigotes proliferate in the insect gut, a compartment full of blood meal. The parasite stores different nutrients, including heme [ 27 ] and lipids, such as cholesterol [ 28 ], in organelles derived from endosomes called reservosomes [ 29 ]. As nutrients become scarce, differentiation of epimastigotes into metacyclic-trypomastigotes is triggered and concomitantly these endosomes are consumed [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

et al. 2015

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Translation initiation has been described as a key step for the control of growth and differentiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the expression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas’ disease, as a transmembrane protein located in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the absence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of proliferative into infective and non-proliferative forms. Parasites lacking TcK2 lose this differentiation capacity and heme is not stored in reserve organelles, remaining in the cytosol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. The augmented level of hydrogen peroxide occurs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi.

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Section: Resultsmentioning

confidence: 99%

A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

et al. 2015

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“…Recently, it has been demonstrated that Leishmania parasites are able to acquire significant amounts of cholesterol from LDL particle endocytosis (Andrade‐Neto et al., ; De Cicco et al., ). This strategy has also been illustrated for other protozoan parasites such as Toxoplasma gondii , Trypanosoma cruzi , and Cryptosporidium parvum , which indicate that cholesterol is widely important for the intracellular survival and replication of pathogens (Coppens, Sinai, & Joiner, ; Ehrenman, Wanyiri, Bhat, Ward, & Coppens, ; Johndrow et al., ; Pereira et al., ; Portugal et al., ). Our experiments using LDL containing 3 H‐labeled cholesteryl linoleate suggest that Leishmania amastigote‐infected cells do not primarily increase the overall cellular uptake of cholesterol, but rather increased retention of cholesterol in the parasite‐containing subcellular fraction is observed when compared to a similarly prepared fraction containing latex beads.…”

Section: Discussionmentioning

confidence: 98%

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

et al. 2017

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Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses.

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“…The parasites were grown in Liver Infusion Tryptose (LIT) medium with 5% Neonatal Bovine Serum [13] at 28°C. Just before performing each experiment, parasites were counted in a Neubauer chamber and used when cultures were at exponential growth (usually 2-6x10 5 parasites/mL) [14]. …”

Section: Methodsmentioning

confidence: 99%

Quantitative Laser Biospeckle Method for the Evaluation of the Activity of Trypanosoma cruzi Using VDRL Plates and Digital Analysis

et al. 2016

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In this paper we report a quantitative laser Biospeckle method using VDRL plates to monitor the activity of Trypanosoma cruzi and the calibration conditions including three image processing algorithms and three programs (ImageJ and two programs designed in this work). Benznidazole was used as a test drug. Variable volume (constant density) and variable density (constant volume) were used for the quantitative evaluation of parasite activity in calibrated wells of the VDRL plate. The desiccation process within the well was monitored as a function of volume and of the activity of the Biospeckle pattern of the parasites as well as the quantitative effect of the surface parasite quantity (proportion of the object’s plane). A statistical analysis was performed with ANOVA, Tukey post hoc and Descriptive Statistics using R and R Commander. Conditions of volume (100μl) and parasite density (2-4x104 parasites/well, in exponential growth phase), assay time (up to 204min), frame number (11 frames), algorithm and program (RCommander/SAGA) for image processing were selected to test the effect of variable concentrations of benznidazole (0.0195 to 20μg/mL / 0.075 to 76.8μM) at various times (1, 61, 128 and 204min) on the activity of the Biospeckle pattern. The flat wells of the VDRL plate were found to be suitable for the quantitative calibration of the activity of Trypanosoma cruzi using the appropriate algorithm and program. Under these conditions, benznidazole produces at 1min an instantaneous effect on the activity of the Biospeckle pattern of T. cruzi, which remains with a similar profile up to 1 hour. A second effect which is dependent on concentrations above 1.25μg/mL and is statistically different from the effect at lower concentrations causes a decrease in the activity of the Biospeckle pattern. This effect is better detected after 1 hour of drug action. This behavior may be explained by an instantaneous effect on a membrane protein of Trypanosoma cruzi that could mediate the translocation of benznidazole. At longer times the effect may possibly be explained by the required transformation of the pro-drug into the active drug.

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Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Cited by 47 publications

References 56 publications

A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

A Membrane-bound eIF2 Alpha Kinase Located in Endosomes Is Regulated by Heme and Controls Differentiation and ROS Levels in Trypanosoma cruzi

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

Quantitative Laser Biospeckle Method for the Evaluation of the Activity of Trypanosoma cruzi Using VDRL Plates and Digital Analysis

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