Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma Cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 11/50631-1)Instituto Nacional de Biologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI)Conselho Nacional de Desenvolvimento Científico e Tecnólogico (CNPq, 470272/2011-2

Influence of caloric restriction on energy metabolism and redox state of Saccharomyces cerevisiae<i/> e Kluyveromyces lactis<i/>

O envelhecimento envolve um progressivo declínio na eficiência metabólica dos sistemas biológicos ao longo do tempo. Embora não possa ser evitado, o envelhecimento pode ter seus fenótipos típicos mitigados em organismos submetidos à restrição calórica, um regime dietético que consiste em uma oferta diminuída de calorias. Ao longo do tempo, a levedura Saccharomyces cerevisiae mostrou-se um importante organismo modelo para o estudo de importantes marcas relacionadas ao envelhecimento, sobretudo por ser responsiva à restrição calórica. Através de uma abordagem do metabolismo energético e do estado de óxido-redução celular, nós temos buscado identificar quais são os fatores imprescindíveis para a exibição do aumento do tempo de vida cronológico dessa levedura. Nós verificamos que defeitos específicos na síntese de nicotinamida adenina dinucleotídeo aumentam a geração mitocondrial de espécies reativas de oxigênio pela enzima dihidrolipoil desidrogenase, porém não suprimem o aumento da do tempo de vida cronológico de S. cerevisiae. Por outro lado, os mutantes dessa levedura irreponsíveis à restrição calórica são aqueles que possuem defeitos no metabolismo aeróbico, mais especificamente na montagem da cadeia de transporte de elétrons. Também verificamos que diferentes mutações em enzimas do ciclo dos ácidos tricarboxílicos alteram a taxa de perda do DNA mitocondrial de S. cerevisiae numa forma dependente da concentração inicial de glicose nos meios de cultura e também do tempo de cultivo. Também observamos que a eficiência energética em S. cerevisiae cultivada sob restrição calórica é aumentada em relação à levedura cultivada em condição controle. Finalmente, também observamos que a morfologia mitocondrial é alterada pelo estado metabólico celular e se correlaciona com a geração de espécies reativas de oxigênio nesse organismo. Assim sendo, em conjunto, esses dados revelam importantes modificações metabólicas e no estado de óxido redução proporcionadas pela restrição calórica e como os fenótipos típicos do envelhecimento podem ser mitigados em S. cerevisiae, assim como quais são os fatores imprescindíveis para a resposta dessa levedura à restrição calórica.Aging involves a progressive decline in metabolic efficiency of biological systems over time. Although it cannot be avoided, aging phenotypes are delayed in organisms undergoing caloric restriction, a dietary regimen consisting of a reduced availability of calories. The yeast Saccharomyces cerevisiae has proved to be an important model organism for studying important characteristics related to aging, and is responsive to caloric restriction. We sought to identify factors essential for increased chronological lifespan in yeast by investigating changes in energy metabolism and redox state. We found that defects in the synthesis of nicotinamide adenine dinucleotide increased mitochondrial generation of reactive oxygen species by the enzyme dihidrolipoil dehydrogenase, but did not suppress the increase in chronological life span. On the other hand, mutants of this yeast which do not respond to caloric restriction are those that have defects in aerobic metabolism, specifically in the assembly of the electron transport chain. We also found that different mutations in enzymes of the citric acid cycle alter the rate of loss of mitochondrial in a manner dependent on the initial concentration of glucose in culture media and culture time. We also observed that energy efficiency in S. cerevisiae grown under caloric restriction is increased compared to yeast grown under control conditions. Finally, we also observed that mitochondrial morphology is altered by the cellular metabolic state and correlates with the generation of reactive oxygen species in this organism. Thus, altogether, these data reveal significant changes in metabolism and redox state promoted by caloric restriction, how phenotypes typical of aging can be prevented in S. cerevisiae, as well as what factors are required for the response of yeast to caloric restriction

cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims: In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods: Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings: cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondria! membrane. Significance: It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients. (C) 2010 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPqPRONEX IIPRONEXFundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS)FAPERGSPROPESQ/UFRGSPROPESQ/UFRGSFAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Financiadora de Estudos e Projetos (FINEP)FINEP[01.06.0842-00]INCT-ENINCT-E

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi

Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi

<div><p>Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.</p></div

Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.CNPq-Brazil (Universal)CNPq-Brazil (INCTV)PRONEXNewton Fund/FAPEMIGFAPESPUniv Fed Minas Gerais, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, SP, BrazilUniv Estadual Campinas, Inst Biol, Dept Bioquim & Biol Tecidual, Campinas, SP, BrazilFiocruz MS, Ctr Pesquisas Rene Rachou, Belo Horizonte, MG, BrazilUniv Fed São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, SP, BrazilWeb of Scienc

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Submitted by Nuzia Santos ([email protected]) on 2019-06-24T16:52:14Z No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-06-24T16:57:35Z (GMT) No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5)Made available in DSpace on 2019-06-24T16:57:35Z (GMT). No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5) Previous issue date: 2018Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG Brazil.Universidade Federal de Minas Gerais. Departamento de Morfologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite