Heme metabolism in Strigomonas culicis: Implications of H2O2 resistance induction and symbiont elimination.

Monoxenous trypanosomatid Strigomonas culicis harbors an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. The impact of H2O2 resistance and symbiont elimination on intracellular heme and Fe2+ availability was analyzed through a comparison of WT strain with both WT H2O2-resistant (WTR) and aposymbiotic (Apo) protozoa. The relative quantification of the heme biosynthetic pathway through label-free parallel reaction monitoring targeted mass spectrometry revealed that H2O2 resistance does not influence the abundance of tryptic peptides. However, the Apo strain showed increased coproporphyrinogen III oxidase and ferrochelatase levels. A putative ferrous iron transporter, homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was identified for the first time. Label-free parallel reaction monitoring targeted mass spectrometry also showed that S. culicis Iron Transporter (ScIT) increased 1.6- and 16.4-fold in WTR and Apo strains compared to WT. Accordingly, antibody-mediated blockage of ScIT decreased by 28.0% and 40.0% intracellular Fe2+concentration in both WTR and Apo strains, whereas no effect was detected in WT. In a heme-depleted medium, adding 10 µM hemin decreased ScIT transcript levels in Apo, whereas 10 µM PPIX, the substrate of ferrochelatase, increased intracellular Fe2+ concentration and ferric iron reduction. Overall, the data suggest mechanisms dependent on de novo heme synthesis (and its substrates) in the Apo strain to overcome reduced heme availability. Given the importance of heme and Fe2+ as cofactors in metabolic pathways, including oxidative phosphorylation and antioxidant systems, this study provides novel mechanistic insights associated with H2O2 resistance in S. culicis.

Is the mitochondrion a promising drug target in trypanosomatids?

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are etiological agents of important neglected tropical diseases, affecting millions of people worldwide, and the drugs available for these diseases present several limitations. Novel efficient and nontoxic drugs are necessary as an alternative to the current chemotherapy. The unique mitochondrion of trypanosomatids and its peculiar features turn this organelle a potential drug target. Several phenotypic studies describe the damage in the parasite mitochondrial ultrastructure, but the molecular target is unknown. Few reports demonstrated the electron transport system (ETS) as a target due to the high similarities to mammalian orthologues, hence ETS is not a good candidate for drug intervention. On the other hand, antioxidant enzymes, such as trypanothione reductase, and an alternative oxidase (AOX) seem to be interesting targets; however no high active inhibitors were developed up to now. Finally, due to the remarkable differences to mammalian machinery, together with the high biological importance for the parasite survival, the mitochondrial import system stands out as a very promising target in trypanosomatids. Archaic translocase of the outer membrane (ATOM) and translocase of the inner membrane (TIM) complexes, which mediate both protein and tRNA import, composed by specific subunits of these parasites, could be excellent candidates, deserving studies focused on the development of specific drugs.

TrypanocidalActivity of Natural Sesquiterpenoids Involves Mitochondrial Dysfunction, ROS Production and Autophagic Phenotype in Trypanosomacruzi.

Chagas disease is a neglected tropical disease that is caused by the protozoan Trypanosomacruzi and represents a serious health problem, especially in Latin America. The clinical treatment of Chagas disease is based on two nitroderivatives that present severe side effects and important limitations. In folk medicine, natural products, including sesquiterpenoids, have been employed for the treatment of different parasitic diseases. In this study, the trypanocidal activity of compounds isolated from the Chilean plants Drimys winteri, Podanthus mitiquiand Maytenus boaria on three T. cruzi evolutive forms (epimastigote, trypomastigote and amastigote) was evaluated. Total extracts and seven isolated sesquiterpenoids were assayed on trypomastigotes and epimastigotes. Polygodial (Pgd) from D. winteri, total extract from P. mitiqui (PmTE) and the germacrane erioflorin (Efr) from P. mitiqui were the most bioactive substances. Pgd, Efr and PmTE also presented strong effects on intracellular amastigotes and low host toxicity. Many ultrastructural effects of these substances, including reservosome disruption, cytosolic vacuolization, autophagic phenotype and mitochondrial swelling (in the case of Pgd), were observed. Flow cytometric analysis demonstrated a reduction in mitochondrial membrane potential in treated epimastigotes and an increase in ROS production and high plasma membrane permeability after treatment with Pgd. The promising trypanocidal activity of these natural sesquiterpenoids may be a good starting point for the development of alternative treatmentsforChagas disease.

Quantitative analysis of proteins secreted by Leishmania (Viannia) braziliensis strains associated to distinct clinical manifestations of American Tegumentary Leishmaniasis.

The role of Leishmania braziliensis in the development of different clinical forms of American Tegumentary Leishmaniasis (ATL) is unclear, but it has been suggested that molecules secreted/released by parasites could modulate the clinical outcome. Here, we analyzed the infection rate and cytokine profile of macrophages pretreated with the secretome of two L. braziliensis strains associated with polar clinical forms of ATL: one associated with localized self-healing cutaneous leishmaniasis (LCL) and other associated with the disseminated form (DL). Besides, we use an iTRAQ-based quantitative proteomics approach to compare the abundance of proteins secreted by those strains. In vitro infection demonstrated that pretreatment with secretome resulted in higher number of infected macrophages, as well as higher number of amastigotes per cell. Additionally, macrophages pretreated with LCL secretome exhibited a proinflammatory profile, whereas those pretreated with the DL one did not. These findings suggest that secretomes made macrophages more susceptible to infection and that molecules secreted by each strain modulate, differentially, the macrophages' cytokine profile. Indeed, proteomics analysis showed that the DL secretome is rich in molecules involved in macrophage deactivation, while is poor in proteins that activate proinflammatory pathways. Together, our results reveal new molecules that may contribute to the infection, persistence and dissemination of the parasite. SIGNIFICANCE: Leishmania braziliensis is associated to localized self-healing cutaneous lesions (LCL), disseminated leishmaniasis (DL), and mucocutaneous lesions (MCL). To understand the role of the parasite in those distinct clinical manifestations we evaluated infection rates and cytokine profiles of macrophages pre-treated with secretomes of two L. braziliensis strains associated with DL and LCL, and quantitatively compared these secretomes. The infection index of macrophages pretreated with the DL secretome was significantly higher than that exhibited by non-treated cells. Interestingly, whereas the LCL secretome stimulated a proinflammatory setting, favoring an effector cell response that would explain the proper resolution of the disease caused by this strain, the DL strain was not able to elicit such response or has mechanisms to prevent this activation. Indeed, DL secretome is rich in peptidases that may deactivate cell pathways crucial for parasite elimination, while is poor in proteins that could activate proinflammatory pathways, favoring parasite infection and persistence.

Glycolytic profile shift and antioxidant triggering in symbiont-free and H2O2-resistant Strigomonas culicis.

During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H2O2-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H2O2 resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.

In-depth quantitative proteomics uncovers specie-specific metabolic programs in Leishmania (Viannia) species.

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.

Is the mitochondrion a promising drug target in trypanosomatids?

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are etiological agents of important neglected tropical diseases, affecting millions of people worldwide, and the drugs available for these diseases present several limitations. Novel efficient and nontoxic drugs are necessary as an alternative to the current chemotherapy. The unique mitochondrion of trypanosomatids and its peculiar features turn this organelle a potential drug target. Several phenotypic studies describe the damage in the parasite mitochondrial ultrastructure, but the molecular target is unknown. Few reports demonstrated the electron transport system (ETS) as a target due to the high similarities to mammalian orthologues, hence ETS is not a good candidate for drug intervention. On the other hand, antioxidant enzymes, such as trypanothione reductase, and an alternative oxidase (AOX) seem to be interesting targets; however no high active inhibitors were developed up to now. Finally, due to the remarkable differences to mammalian machinery, together with the high biological importance for the parasite survival, the mitochondrial import system stands out as a very promising target in trypanosomatids. Archaic translocase of the outer membrane (ATOM) and translocase of the inner membrane (TIM) complexes, which mediate both protein and tRNA import, composed by specific subunits of these parasites, could be excellent candidates, deserving studies focused on the development of specific drugs.

Hydrogen peroxide resistance in Strigomonas culicis: Effects on mitochondrial functionality and Aedes aegypti interaction.

Reactive oxygen species (ROS) are toxic molecules involved in several biological processes such as cellular signaling, proliferation, differentiation and cell death. Adaptations to oxidative environments are crucial for the success of the colonization of insects by protozoa. Strigomonas culicis is a monoxenic trypanosomatid found in the midgut of mosquitoes and presenting a life cycle restricted to the epimastigote form. Among S. culicis peculiarities, there is an endosymbiotic bacterium in the cytoplasm, which completes essential biosynthetic routes of the host cell and may represent an intermediary evolutive step in organelle origin, thus constituting an interesting model for evolutive researches. In this work, we induced ROS resistance in wild type S. culicis epimastigotes by the incubation with increasing concentrations of hydrogen peroxide (H2O2), and compared the oxidative and energetic metabolisms among wild type, wild type-H2O2 resistant and aposymbiotic strains. Resistant protozoa were less sensitive to the oxidative challenge and more dependent on oxidative phosphorylation, which was demonstrated by higher oxygen consumption and mitochondrial membrane potential, increased activity of complexes II-III and IV, increased complex II gene expression and higher ATP production. Furthermore, the wild type-H2O2 resistant strain produced reduced ROS levels and showed lower lipid peroxidation, as well as an increase in gene expression of antioxidant enzymes and thiol-dependent peroxidase activity. On the other hand, the aposymbiotic strain showed impaired mitochondrial function, higher H2O2 production and deficient antioxidant response. The induction of H2O2 resistance also led to a remarkable increase in Aedes aegypti midgut binding in vitro and colonization in vivo, indicating that both the pro-oxidant environment in the mosquito gut and the oxidative stress susceptibility regulate S. culicis population in invertebrates.

Mecanismos de resistência às espécies reativas em tripanosomatídeos: contribuição do metabolismo energético e oxidativo

As doenças tropicais negligenciadas ameaçam a vida de mais de um bilhão de pessoas em todo o mundo, estando, aproximadamente, meio bilhão sob risco de infecção por parasitos da família Trypanosomatidae. A colonização de ambientes diferentes em seus hospedeiros faz com que os tripanosomatídeos sejam constantemente submetidos a situações de estresse, dentre as quais a presença de espécies reativas de oxigênio (ROS) e nitrogênio (RNS); exigindo o remodelamento metabólico para garantir a sobrevivência do parasito em ambientes "hostis". Algumas cepas de Leishmania (Viannia) braziliensis são capazes de burlar esses mecanismos microbicidas, exibindo um perfil de resistência natural ao óxido nítrico (•NO). Este já foi associado ao aumento na quantidade e gravidade das lesões cutâneas, além de ter sido frequentemente correlacionado à refratariedade ao tratamento com antimonial. De forma semelhante, a resistência induzida ao peróxido de hidrogênio (H2O2) também aumentou a capacidade infectiva de Strigomonas culicis, um tripanosomatídeo monoxênico, em seu hospedeiro natural Aedes aegypti. Juntos, esse dados sugerem que parasitos resistentes às espécies reativas são dotados de mecanismos específicos de sobrevivência e persistência frente aos desafios oxidativos e nitrosativos; dessa forma, investigamos, através de técnicas proteômicas e bioquímicas, diferenças intrínsecas entre as cepas NO- e H2O2-resistentes e seus pares susceptíveis. Nossos dados apontaram que tanto a resistência natural ao •NO, quanto à induzida ao H2O2, elevaram a expressão e atividade de vias antioxidantes independentes do sistema tripanotiona/tripanotiona redutase (TR), principalmente aquelas relacionadas à via de biossíntese da glutationa e a ascorbato peroxidase. Adicionalmente, também observamos um aumento na abundância de proteínas da via das pentoses fosfato como um mecanismo de resposta às situações de estresse, podendo este estar relacionado à manutenção dos níveis de NADPH no protozoário. A regulação positiva do consumo de glicose e da capacidade redox identificada na cepa NO-resistente de L. (V.) braziliensis pode explicar as altas taxas de infecção detectadas em macrófagos peritoneais murinos, justificando a sobrevivência desses parasitos no ambiente pró-oxidante do vacúolo parasitóforo. Já a infecção de A. aegypti pela cepa H2O2-resistente de S. culicis exacerbou a produção de ROS pelo hospedeiro, através do aumento da atividade DUOX e inibição da catalase, gerando danos significativos na aptidão reprodutiva das fêmeas infectadas. As vias bioenergéticas e antioxidantes dos tripanosomatídeos também representam um excelente alvo para intervenção medicamentosa. Nesse contexto, também avaliamos o papel de derivados da -lapachona, uma molécula com função pró-oxidante bem descrita, no funcionamento e na produção de ROS mitocondrial pelo T. cruzi. Análises mecanísticas apontaram o comprometimento direto da cadeia respiratória pelos compostos N2, N3 e N4, levando ao escape de elétrons e a formação de espécies tóxicas para o parasito. Interessantemente, após 24 h de tratamento com N4, uma restauração parcial da fisiologia mitocondrial foi observada, estando esta relacionada à atividade da TR, que aumentou de forma tempo-dependente e concomitantemente ao estresse oxidativo. Sendo assim, a exposição de tripanosomatídeos dixênicos e monoxênicos à diferentes situações de estresse oxidativo e nitrosativo desencadeia mecanismos de sobrevivência relacionados à exacerbação das vias antioxidantes e ao remodelamento energético

Avaliação do metabolismo oxidativo e energético de Strigomonas culicis e suas implicações na interação com o hospedeiro

Strigomonas culicis é um protozoário monoxênico encontrado no intestino médio de vários mosquitos, apresentando um ciclo de vida restrito à forma epimastigota. Dentre as suas peculiaridades, existe a presença de uma bactéria endossimbiótica, cujo papel biológico envolve a captação de heme e ferro para o protozoário, moléculas envolvidas no metabolismo energético e oxidativo. Apesar da colonização do intestino de insetos hematófagos, ambiente rico em espécies reativas de oxigênio, uma avaliação detalhada dos mecanismos antioxidantes desses protozoários ainda não foi realizada. Neste trabalho, avaliamos o metabolismo oxidativo e energético de S. culicis comparando três diferentes cepas: selvagem (WT), apossimbiótica (Apo) e selvagem H2O2-resistente (WTR). Apo foi mais susceptível ao estresse oxidativo, apresentando uma maior captação de glicose e fosforilação oxidativa reduzida, além de apresentar atividade antioxidante menos eficiente e expressão gênica aumentada de três isoformas da triparedoxina. Por outro lado, WT apresentou maior resistência ao estresse oxidativo, especialmente a altos níveis de H2O2, além de uma dependência maior da mitocôndria para a obtenção de energia. WTR apresentou uma maior resistência ao desafio oxidativo e maior dependência da fosforilação oxidativa, demonstrado através do maior consumo de oxigênio e do potencial de membrana mitocondrial, do aumento da atividade dos complexos II-III e IV além da alta produção de ATP, sendo ainda observado um aumento da expressão gênica do complexo II Esta cepa ainda produziu níveis reduzidos de ROS e de peroxidação lipídica em relação às demais, com o aumento na expressão gênica de uma das isoformas de triparedoxina. Apesar das alterações fisiológicas, não foram encontradas alterações ultraestruturais na WTR, inclusive na mitocôndria. A indução de resistência também levou a uma maior colonização do intestino médio de Aedes aegypti ex vivo e in vivo, o que reforça a hipótese de que o ambiente pro-oxidante no intestino do mosquito regula a população de S. culicis, dado corroborado pelo aumento da colonização do intestino pelas três cepas do protozoário após a alimentação de A. aegypti com ascorbato ad libitum. WTR ainda apresentou um aumento na adesão a macrófagos peritoneais murinos, demonstrando a influência da resistência ao estresse oxidativo também na interação com células de mamíferos. Desta forma, as estratégias metabólicas e antioxidantes de S. culicis começaram a ser descritas bem como o papel do endossimbionte no processo, o que contribui para a compreensão dos mecanismos de resistência e persistência do protozoário em mamíferos, incluindo o homem.