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

Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation

The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake

Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite’s replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE) with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport), which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi.Fil: Merli, Marcelo Luciano. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Pagura, Lucas. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Hernández, Josefina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Barisón, María Julia. Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. Laboratory of Biochemistry of Tryps (LaBTryps); Brasil.Fil: Pral, Elizabeth M. F. Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. Laboratory of Biochemistry of Tryps (LaBTryps); Brasil.Fil: Silber, Ariel M. Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. Laboratory of Biochemistry of Tryps (LaBTryps); Brasil.Fil: Cricco, Julia Alejandra. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina

Targeting L-Proline uptake as new strategy for anti-chagas drug development

L-Proline is an important amino acid for the pathogenic protists belonging to Trypanosoma and Leishmania genera. In Trypanosoma cruzi, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses. In this study, we explore the L-Proline uptake as a chemotherapeutic target for T. cruzi. Novel inhibitors have been proposed containing the amino acid with a linker and a variable region able to block the transporter. A series of sixteen 1,2,3-triazolyl-proline derivatives have been prepared for in vitro screening against T. cruzi epimastigotes and proline uptake assays. We successfully obtained inhibitors that interfere with the amino acid internalization, which validated our design targeting the metabolite’s transport. The presented structures are one of few examples of amino acid transporter inhibitors. The unprecedent application of this strategy on the development of new chemotherapy against Chagas disease, opens a new horizon on antiparasitic drug development against parasitic diseases and other pathologies.Para citar este articulo: Fargnoli L, Panozzo-Zénere EA, Pagura L, Barisón MJ, Cricco JA, Silber AM and Labadie GR (2020) Targeting L-Proline Uptake as New Strategy for Anti-chagas Drug Development. Front. Chem. 8:696. doi: 10.3389/fchem.2020.00696Fil: Fargnoli, Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario (IQUIR -CONICET); Argentina.Fil: Panozzo Zénere, Esteban Andrés. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario (IQUIR -CONICET); Argentina.Fil: Pagura, Lucas. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Barisón, María Julia. Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. Laboratory of Biochemistry of Tryps (LaBTryps); Brazil.Fil: Cricco, Julia Alejandra. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Silber, Ariel M. Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. Laboratory of Biochemistry of Tryps (LaBTryps); Brazil.Fil: Labadie, Guillermo Roberto. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario (IQUIR -CONICET); Argentina.Fil: Labadie, Guillermo Roberto. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica; Argentina

New L-proline uptake inhibitors with anti-Trypanosoma cruzi activity

L-Proline is an important amino acid for the pathogenic protists belonging to Trypanosoma and Leishmania genera. In Trypanosoma cruzi, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses, including nutritional and osmotic as well as oxidative imbalance. In this study, we explore the L-Proline uptake as a chemotherapeutic target for T. cruzi. For this, we propose a novel rational to design inhibitors containing this amino acid as a recognizable motif. This rational consists of conjugating the amino acid (proline in this case) to a linker and a variable region able to block the transporter. We obtained a series of sixteen 1,2,3-triazolyl-proline derivatives through alkylation and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) for in vitro screening against T. cruzi epimastigotes, trypanocidal activity and proline uptake. We successfully obtained inhibitors that are able to interfere with the amino acid uptake, which validated the first example of a rationally designed chemotherapeutic agent targeting a metabolite\u27s transport. Additionally, we designed and prepared fluorescent analogues of the inhibitors that were successfully taken up by T. cruzi, allowing following up their intracellular fate. In conclusion, we successfully designed and produced a series of metabolite uptake inhibitors. This is one of few examples of rationally designed amino acid transporter inhibitor, being the first case where the strategy is applied on the development of chemotherapy against Chagas disease. This unprecedented development is remarkable having in mind that only a small percent of the metabolite transporters has been studied at the structural and/or molecular level

Effect of Memantine on the intracellular cycle of <i>Trypanosoma cruzi</i>.

<p>Panel A: viability of CHO-K₁ cells treated with different concentrations of Memantine (range 50 µM to 1 mM). Viability was assessed by MTT assay. Panel B: effect on the infectivity of trypomastigotes treated only during the period of infection (50–400 µM). Panel C: effect of treatment after invasion of parasites in CHO-K₁ cells (5–300 µM). Panel D: effect of Memantine on intracellular stages. Cells were treated at different stages with 31 µM Memantine (IC₅₀ value): <b>T</b> (trypomastigote cell invasion), <b>A</b> (amastigote) and <b>IE</b> (intracellular epimastigote-like) stages. In all experiments, we evaluated the burst of trypomastigotes on the fifth day post-infection by counting parasites in a Neubauer chamber. Tukey test: *: p<0.05; **: p<0.01; ***: p<0.001.</p

Chemical structures of Memantine, Amantadine and MK-801.

<p>Chemical structures of Memantine, Amantadine and MK-801.</p

Analysis of extracellular phosphatidylserine exposure.

<p>Panel A: untreated parasites. Panel B: treated parasites. The parasites were treated with Memantine (172.6 µM) or not treated (control) for 4 days. After this period, the parasites were labeled with propidium iodide (PI) and annexin V and analyzed by flow cytometry. Panel C: quantitative analysis of three independent experiments (<i>T</i> test: ***: p<0.001).</p

Forward and side scattering values for epimastigotes treated or not with Memantine.

1<p>Geometrical mean of scatter values of epimastigotes treated or not with 172.6 µM Memantine.</p

TcHTE promotes heme uptake in <i>hem1</i>Δ yeast cells.

<p>Spot growth assay of <i>S</i>. <i>cerevisiae hem1</i>Δ transformed with the p426.MET25 or the vector carrying <i>TcHTE</i>.<i>6HIS</i> or <i>TcHTE</i>.<i>6HIS-GFP</i> constructs. 7 μL of four serial dilutions from the initial cultures of OD = 2 were plated in a solid SC medium with Glucose (A) or Glycerol-Ethanol (B) as a carbon source, and different hemin concentrations, or δ-ALA as control. Western blot of total cell extracts of <i>S</i>. <i>cerevisiae</i> transformed with vector carrying <i>TcHTE</i>.<i>6HIS-GFP</i> with anti-GFP or anti-His antibodies (C). Confocal microscopy images of <i>S</i>. <i>cerevisiae hem1</i>Δ transformed with a vector carrying <i>TcHTE</i>.<i>6HIS-GFP</i> growth in SC medium with Glucose and 0.5 μM hemin (D).</p