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

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- 14 C]-palmitate can be taken up from the medium, leading to CO 2 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.Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomesInteractions métaboliques entre les adipocytes et les trypanosomes, un nouveau paradigme pour les trypanosomosesAlliance française contre les maladies parasitaire

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly

Characterization of fatty acid metabolism in Trypanosoma cruzi.

Está bem estabelecido que o metabolismo do T. cruzi, agente etiológico da doença de Chagas, é baseado principalmente no consumo de glicose e/ou aminoácidos, dependendo da disponibilidade desses substratos no ambiente. Neste trabalho foi demonstrada a importância do metabolismo de ácidos graxos, principalmente para as formas do parasita que estão presentes no inseto vetor. T. cruzi é capaz de incorporar ácidos graxos a partir do meio externo e metabolizar esses substratos, gerando CO2. Mostramos que a degradação de ácidos graxos em T. cruzi é operativa nas formas epimastigotas (formas proliferativas e não infectivas presentes no inseto vetor), porém sua ativação depende da disponibilidade de glicose no meio. À medida que diminui a concentração de glicose no meio aumenta a dependência da utilização de ácidos graxos como fonte de energia. Essa regulação é concomitante com o aumento da atividade da Carnitina palmitoiltransferase 1 (CPT1), principal reguladora do processo de degradação de ácidos graxos. Também foi observado que o parasita pode realizar síntese de novo de ácidos graxos a partir de malonil-CoA, no entanto, a atividade da Acetil-CoA carboxilase (ACC) também está condicionada à concentração de glicose, sendo mais ativa no início da curva de proliferação. O tratamento de formas epimastigotas de T. cruzi com Etomoxir, inibidor de CPT1, interfere na proliferação dos parasitas, impedindo a ativação da beta-oxidação quando os níveis de glicose no meio estão mais baixos. Etomoxir também diminui a resistência dos parasitas ao estresse nutricional severo, fazendo com que ácidos graxos se acumulem em inclusões lipídicas, sustentando a ideia de que esses parasitas podem utilizar conteúdo lipídico na forma de triacilgliceróis como reserva energética. Nesse sentido, foi caracterizada uma das enzimas críticas no metabolismo de lipídeos, a Acil-CoA desidrogenase (ACAD), responsável pelo primeiro passo da beta-oxidação. O gene de T. cruzi que codifica para a ACAD com características de ACAD para substratos de cadeia média (C8:0 C16:0) foi expresso em Escherichia coli e a ACAD recombinante resultante (TcACAD) foi caracterizada. Notavelmente possui amplo espectro de substratos, capaz de reconhecer substratos de cadeia curta e longa e até mesmo substratos com ramificação, derivados da via de degradação de aminoácidos de cadeia ramificada. A TcACAD está localizada na mitocôndria do parasita e é expressa diferencialmente ao longo do ciclo de vida do parasita, sendo mais ativa nas formas epimastigotas intracelulares, presentes no hospedeiro mamífero. A TcACAD possui uma atividade parcial de acil-CoA oxidase, gerando H2O2 na mitocôndria. Finalmente, também foi mostrado que a beta-oxidação é um processo metabólico importante para a diferenciação. Diante do exposto, conclui-se que o metabolismo de ácidos graxos contribui para o metabolismo energético do parasita.It is well established that the metabolism of T. cruzi, the etiologic agent of Chagas\' disease, is based mainly on the consumption of glucose and/or amino acids, depending on the availability of these substrates in the environment. In this work the importance of fatty acid metabolism was demonstrated, especially for the forms of the parasite that are present in the insect vector. T. cruzi is able to incorporate fatty acids from the external environment and metabolize these substrates, generating CO2. We show that fatty acid degradation in T. cruzi is operative in the epimastigote forms (proliferative and noninfective forms present in the insect vector), but its activation depends on the availability of glucose in the medium. When the glucose concentration in the medium decreases the dependence of the use of fatty acids as energy source increases. This regulation is concomitant with the increase in the activity of Carnitine palmitoyltransferase 1 (CPT1), the main regulator of the fatty acid degradation process. It was also observed that the parasite could perform de novo synthesis of fatty acids from malonyl- CoA, however, the activity of Acetyl-CoA carboxylase (ACC) is also conditioned to glucose concentration, being more active at the beginning of the curve of proliferation. The treatment of epimastigotes forms of T. cruzi with Etomoxir, inhibitor of CPT1, interferes in the proliferation of the parasites, preventing the activation of the beta-oxidation when the glucose levels in the middle are lower. Etomoxir also decreases parasite resistance to severe nutritional stress, causing fatty acids to accumulate in lipid inclusions, supporting the idea that these parasites can use lipid content in the form of triacylglycerols as an energy reserve. In this sense, one of the critical enzymes in lipid metabolism, Acyl-CoA dehydrogenase (ACAD), was responsible for the first step of beta-oxidation. The T. cruzi gene encoding ACAD with ACAD characteristics for medium chain substrates (C8: 0-C16: 0) was expressed in Escherichia coli and the resulting recombinant ACAD (TcACAD) was characterized. Notably, it has a broad spectrum of substrates capable of recognizing short and long chain substrates and even branched substrates derived from the branched chain amino acid degradation pathway. TcACAD is located in the mitochondria of the parasite and is differentially expressed throughout the life cycle of the parasite, being more active in the intracellular epimastigot forms present in the mammalian host. TcACAD has a partial activity of acyl-CoA oxidase, generating H2O2 in mitochondria. Finally, it has also been shown that beta-oxidation is an important metabolic process for differentiation. In view of the above, it is concluded that the metabolism of fatty acids contributes to the energetic metabolism of the parasite

UM ESTUDO SOBRE A “TIC” E O ENSINO DA QUÍMICA

Atualmente, as novas tecnologias de comunicação e informação estão proporcionando diferentes formas de expressão para a população mundial, em particular para os jovens. Contudo, é necessário que façamos da tecnologia uma aliada a favor da humanidade, usando principalmente como processo de ensino-aprendizagem nas escolas. Neste contexto, a TIC funciona como um recurso na inserção de um novo método de ensino, despertando no aluno o interesse dos assuntos abordados, muitas vezes em sala de aula. Diante disso, as TICs podem ajudar a uma melhor compreensão de algumas disciplinas. Assim, esse trabalho faz um estudo de quanto a TIC pode ser importante no processo de ensino-aprendizagem na disciplina de química, durante o Ensino Médio. Para a realização do presente trabalho foi realizado um estudo bibliográfico e em sites confiáveis de pesquisa</p

Protein control of membrane and organelle dynamics: insights from the divergent eukaryote Toxoplasma gondii

Integral membrane protein complexes control key cellular functions in eukaryotes by defining membrane-bound spaces within organelles and mediating inter-organelles contacts. Despite the critical role of membrane complexes in cell biology, most of our knowledge is from a handful of model systems, primarily yeast and mammals, while a full functional and evolutionary understanding remains incomplete without the perspective from a broad range of divergent organisms. Apicomplexan parasites are single-cell eukaryotes whose survival depends on organelle compartmentalisation and communication. Studies of a model apicomplexan, Toxoplasma gondii, reveal unexpected divergence in the composition and function of complexes previously considered broadly conserved, such as the mitochondrial ATP synthase and the tethers mediating ER–mitochondria membrane contact sites. Thus, Toxoplasma joins the repertoire of divergent model eukaryotes whose research completes our understanding of fundamental cell biology

Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

BackgroundLeishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies.Methods/principal findingsPhagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79.Conclusions/significanceProteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species