Measurement of Energy States of the Trypanosomatid Mitochondrion

Alencar, Mayke Bezerra; Girard, Richard M. B. M.; Silber, Ariel Mariano

doi:10.1007/978-1-0716-0294-2_39

Cited by 6 publications

(7 citation statements)

References 26 publications

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“…We then measured the leak respiration by inhibiting the ATP synthase with oligomycin A. Finally, to measure the maximum capacity of the electron transport system (ETS), we used the uncoupler FCCP [21]. Our results demonstrate that compared to no treatment, ETO treatment diminishes the rate of basal oxygen consumption, the leak respiration and the ETS capacity.…”

Section: Resultsmentioning

confidence: 99%

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Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages ofTrypanosoma cruzi

Souza

Damasceno

Marsiccobetre

et al. 2021

Preprint

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During its complex life cycle, Trypanosoma cruzi colonizes different niches in its insect and mammalian hosts. This characteristic determined the types of parasites that adapted to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. These T. cruzi stages could be exposed to fatty acids originating from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta-oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to feed other anabolic pathways such as the de novo biosynthesis of fatty acids.Author SummaryTrypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

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Section: Resultsmentioning

confidence: 99%

“…Oligomycin A (0.5 µg/mL) and FCCP (0.5 µM) were sequentially added to measure the optimal non-coupled respiration and the respiration leak state, respectively. The data were recorded and treated using DatLab 7 software [15,16,21].…”

Section: Co 2 Production From Oxidisable Carbon Sourcesmentioning

confidence: 99%

Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages ofTrypanosoma cruzi

Souza

Damasceno

Marsiccobetre

et al. 2021

Preprint

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“…We then measured the leak respiration by inhibiting the ATP synthase with oligomycin A. Finally, to measure the maximum capacity of the electron transport system (ETS), we used the uncoupler FCCP [27]. Our results demonstrate that compared to no treatment, ETO treatment diminishes the rate of basal O 2 consumption, the leak respiration and the ETS capacity (Fig 10C).…”

Section: Plos Pathogensmentioning

confidence: 86%

“…Oligomycin A (0.5 μg/mL) and FCCP (0.5 μM) were sequentially added to measure the optimal non-coupled respiration and the respiration leak state, respectively. The data were recorded and treated using DatLab 7 software [15,16,27].…”

Section: Fatty Acid Oxidation Assaysmentioning

confidence: 99%

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi

et al. 2021

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Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.

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“…Then, 2.2 μM, 4.4 μM or 6.6 μM A6 (1x, 2x, and 3x the IC 50 concentrations) were sequentially added. H 2 O 2 production was quantified by following the reduction of AmR which emits fluorescence in its reduced state as described in detail [ 32 ]. Experiments in each condition were run in three biological independent replicas and data were recorded and treated by using DatLab 7 software.…”

Section: Methodsmentioning

confidence: 99%

An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection

et al. 2021

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Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6–8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 “chimeric” molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 μM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 μM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

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Measurement of Energy States of the Trypanosomatid Mitochondrion

Cited by 6 publications

References 26 publications

Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages ofTrypanosoma cruzi

Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages ofTrypanosoma cruzi

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi

An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection

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