Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that threaten genome integrity. To address DSBs, eukaryotic cells of model organisms have evolved a complex network of cellular pathways that are able to detect DNA damage, activate a checkpoint response to delay cell cycle progression, recruit the proper repair machinery, and resume the cell cycle once the DNA damage is repaired. Cell cycle checkpoints are primarily regulated by the apical kinases ATR and ATM, which are conserved throughout the eukaryotic kingdom. Trypanosoma brucei is a divergent pathogenic protozoan parasite that causes human African trypanosomiasis (HAT), a neglected disease that can be fatal when left untreated. The proper signaling and accuracy of DNA repair is fundamental to T. brucei not only to ensure parasite survival after genotoxic stress but also because DSBs are involved in the process of generating antigenic variations used by this parasite to evade the host immune system. DSBs trigger a strong DNA damage response and efficient repair process in T. brucei, but it is unclear how these processes are coordinated. Here, by knocking down ATR in T. brucei using two different approaches (conditional RNAi and an ATR inhibitor), we show that ATR is required to mediate intra-S and partial G1/S checkpoint responses. ATR is also involved in replication fork stalling, is critical for H2A histone phosphorylation in a small group of cells and is necessary for the recruitment and upregulation of the HR-mediated DNA repair protein RAD51 after ionizing radiation (IR) induces DSBs. In summary, this work shows that apical ATR kinase plays a central role in signal transduction and is critical for orchestrating the DNA damage response in T. brucei.

ATR kinase is a crucial player mediating the DNA damage response in Trypanosoma brucei

DNA double-strand breaks (DSBs) are among the most deleterious lesions that threaten genome integrity. To address DSBs, eukaryotic cells of model organisms have evolved a complex network of cellular pathways that are able to detect DNA damage, activate a checkpoint response to delay cell cycle progression, recruit the proper repair machinery, and resume the cell cycle once the DNA damage is repaired. Cell cycle checkpoints are primarily regulated by the apical kinases ATR and ATM, which are conserved throughout the eukaryotic kingdom. Trypanosoma brucei is a divergent pathogenic protozoan parasite that causes human African trypanosomiasis (HAT), a neglected disease that can be fatal when left untreated. The proper signaling and accuracy of DNA repair is fundamental to T. brucei not only to ensure parasite survival after genotoxic stress but also because DSBs are involved in the process of generating antigenic variations used by this parasite to evade the host immune system. DSBs trigger a strong DNA damage response and efficient repair process in T. brucei, but it is unclear how these processes are coordinated. Here, by knocking down ATR in T. brucei using two different approaches (conditional RNAi and an ATR inhibitor), we show that ATR is required to mediate intra-S and partial G1/S checkpoint responses. ATR is also involved in replication fork stalling, is critical for H2A histone phosphorylation in a small group of cells and is necessary for the recruitment and upregulation of the HR-mediated DNA repair protein RAD51 after ionizing radiation (IR) induces DSBs. In summary, this work shows that apical ATR kinase plays a central role in signal transduction and is critical for orchestrating the DNA damage response in T. brucei.

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation.

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.

Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2).

BACKGROUND: Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2. METHODS/PRINCIPAL FINDINGS: The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms. CONCLUSIONS AND SIGNIFICANCE: This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.

Phosphorylation of eIF2α on Threonine 169 is not required for Trypanosoma brucei cell cycle arrest during differentiation.

The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating 'slender' bloodstream forms differentiate to non-dividing 'stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.