Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi

Among the many environmental challenges the parasiteTrypanosoma cruzihas to overcome to complete its lifecycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounterdistinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released fromhemoglobin degradation in the triatomine´s midgut. Also, the redox status of the surroundings functions as asignal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular secondmessengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellularprocesses accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation,differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypicalpharmacological properties and some have been involved in key processes for the survival of the parasites, whichvalidates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processesaccording to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMPanalogs, while in trypomastigotes an increase in intracellular cAMP doesn´t seem to aid in this response, althoughit does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, weshow that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epi-mastigotes response to oxidative stress.Fil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Signal Transduction Pathways as Therapeutic Target for Chagas Disease

Trypanosomatids are a group of flagellated unicellular eukaryotes, which cause serious human diseases including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.) and Leishmaniasis (Leishmania spp.). The second messenger cAMP is involved in numerous and fundamental processes in these parasites including differentiation between stages, proliferation, osmoregulation, oxidative stress and quorum sensing. Interestingly, its signaling pathway is quite different from that of mammals, including structurally different adenylyl cyclases, the shortage of orthologous effector proteins and the absence of G-protein-coupled-receptors, among others. These characteristics make the proteins involved in these transduction pathways good candidates for therapeutic targets. However, the identification of new unknown druggable targets involves extensive research time and is economically very expensive, making the transition from basic research to the clinical phase difficult. Trypanosomatid PDEs have characteristic binding pockets that allow for a differential inhibition from their human orthologs. Modification of human use approved drugs to turn them into trypanocidal treatments could lead to more effective therapies and shorter lab to counter top transition times and lower costs. In view of the fact that kinetoplastid PDEs are highly conserved with their mammalian counterparts, and since there are already numerous drugs on the market against human PDEs, the drug repositioning approach is highly promising. The development of new technologies, higher government and industrial involvement and more scientists committed to basic investigation, are the key to ultimately find an effective treatment and cure for de the neglected tropical diseases.Fil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

TcAMPK: A cellular energy homeostasis hub regulator with unique characteristics in Trypanosoma cruzi

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to different stresses in many organisms. During the transition between the mammalian host and the insect vector, Trypanosoma cruzi, the causative agent of Chagas disease, faces different types of environmental fluctuations, all of which prompt the parasite to remodel its metabolism to adapt, survive and differentiate into the next stages of its life cycle. Recently, it was shown that Trypanosoma brucei AMPK is involved in the differentiation from the bloodstream slender to stumpy stage and in surface protein expression changes in response to nutritional stress. This underscores the relevance of AMPK for parasite life cycle progression. We identified four genes for the AMPK subunits of T. cruzi (α1 and α2 catalytic subunits, β and γ regulatory subunits). The β and γ subunits are largely conserved in their domain structure relative to the mammalian orthologs. However, the alpha subunits show significant sequence, structure and evolutionary differences from the human counterparts. The presence of these subunits in epimastigotes was confirmed by several techniques. TcAMPKα1 over-expressing epimastigotes showed a lower growth rate in basal culture conditions compared to the control, while α2 over-expression had the opposite effect. We found there is upregulation of AMPK activity under starvation, which is prevented if glucose is present in the culture medium. Each of these subunits could complement the ?glucose dependent? phenotype of S. cerevisiae conditional mutants lacking the respective subunit of the AMPK ortholog SNF1. Starvation and Monodansylcadaverine incorporation assays with AMPKα over-expressing parasites also showed a possible role of AMPK in autophagy. Finally, we explore the pathways in which TcAMPK may be involved using in silico analysis of putative substrates. Our results open the door to the study of the AMPK signaling pathway in Trypanosoma cruzi.Fil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Genta, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXXII Reunión Anual de la Sociedad Argentina de ProtozoologíaCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Protozoologí

TcAMPK: A cellular energy homeostasis hub regulator with unique characteristics in Trypanosoma cruzi

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved inmaintaining energy homeostasis in response to different stresses. During the transitionbetween the mammalian host and the insect vector, Trypanosoma cruzi (Chagasdisease), faces different environmental fluctuations, all of which prompt the parasite toremodel its metabolism to adapt and survive. Recently, it was shown that Trypanosomabrucei AMPK is involved in the differentiation process and in surface protein expressionchanges in response to nutritional stress.We identified four genes for the AMPK subunits of T. cruzi (α1 and α2 catalytic subunits,β and γ regulatory subunits). The β and γ subunits are largely conserved in their domainstructure. However, the alpha subunits show significant sequence, structure andevolutionary differences from the human counterparts. The presence of these subunits inT. cruzi epimastigotes was confirmed by several experimental approaches. TcAMPKαsubunits over-expression in epimastigotes showed opposite effects on growth rate. AMPKactivity is also upregulated in epimastigote under starvation. Each of these subunitscould complement the ?glucose dependent? phenotype of S. cerevisiae conditionalmutants lacking SNF1. Starvation assays with AMPKα over-expressing parasites alsoshowed a role of AMPK in autophagy. Our results confirm the presence of a functionalAMPK orthologue in T. cruzi. We also explore the pathways in which TcAMPK may beinvolved using in silico analysis of putative substrates.Fil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Genta, Patricio D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaParasitravaganza 2021SmithfieldAustraliaThe Australian Society for Parasitolog

Functional roles of AMP-activated protein kinase (AMPK) complexes containing TcAMPKα1 or TcAMPKα2 in energy homeostasis regulation and cell culture progression in Trypanosoma cruzi

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to different stresses in many organisms. During the transition between the mammalian host and the insect vector, Trypanosoma cruzi, the causative agent of Chagas disease, faces different types of environmental fluctuations, all of which prompt the parasite to remodel its metabolism. Recently, it was shown that Trypanosoma brucei AMPK is involved in the differentiation from the bloodstream slender to stumpy stage and in surface protein expression changes in response to nutritional stress. This underscores the relevance of AMPK for parasite life cycle progression.We identified four candidate genes for the AMPK subunits of T. cruzi (alpha1 and alpha2 catalytic subunits, beta and gamma regulatory subunits). The beta and gamma subunits are largely conserved in their domain structure relative to the mammalian orthologs. However, the alpha subunits show significant sequence and structure differences from the human counterparts. The presence of these subunits in T. cruzi epimastigotes was confirmed by RT-PCR, Western blot using a phospho-AMPKa specific antibody, mass spectrometry and by kinase activity assays using the specific AMPK substrate SAMS. TcAMPKa1 over-expressing epimastigotes showed a lower growth rate in basal culture conditions compared to the control. On the other hand, alpha2 over-expression had the opposite effect. Additionally, we observed upregulation of AMPK activity under epimastigote starvation, and that dorsomorphin, a specific AMPK inhibitor, also inhibits T. cruzi AMPK. Moreover, each of these subunits could complement S. cerevisiae conditional mutants lacking the respective subunit of the AMPK ortholog SNF1. Finally, starving assays with AMPKa over-expressing parasites also showed a possible role of AMPK in autophagy. Overall, our results show for the first time, the presence of a functional AMPK orthologue in Trypanosoma cruzi.Fil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Genta, Patricio D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaLXIV Annual Meeting of Sociedad Argentina de Investigación Clínica (SAIC) ;LI Annual Meeting of Asociación Argentina de Farmacología Experimental (SAFE); XXI Annual Meeting of Sociedad Argentina de Biología (SAB); XXXI Annual Meeting of Sociedad Argentina de Protozoología (SAP); IX Annual Meeting of Asociación Argentina de Nanomedicinas (NANOMED-ar); VI Regional Scientific Meeting of Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio (AACyTAL)Mar del PlataArgentinaSociedad Argentina de ProtozoologíaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentaSociedad Argentina de BiologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de Laboratori

Análisis evolutivo de las isoformas de la AMPKα presente en Trypanosoma cruzi

La quinasa de proteínas dependiente de AMP (AMPK) es una enzima clave en el sensado energético que cuenta con una subunidad catalítica (AMPKα) y dos subunidades regulatorias (AMPKβ y APMKγ), y según la especie puede hallarse más de una isoforma. Trypanosoma cruzi, parásito unicelular con un ciclo de vida complejo, conserva dicho complejo presentando dos isoformas de la AMPKα (TcAMPKα1 y TcAMPKα2) y una única isoforma para cada una de las otras dos subunidades. La mayoría de estudios filogenéticos que involucran a estos organismos se suelen incluir otros eucariotas evolutivamente distantes que generan fenómenos que le restan fiabilidad a las relaciones establecidas debido a las grandes distancias filogenéticas. En el presente trabajo realizamos estudios evolutivos comparativos para cada una de las AMPKα incluyendo organismos pertenecientes al grupo de los Tritryps. A partir de estos primeros análisis observamos una alta conservación de la secuencia de cada isoforma dentro de cada especie. También se observó que para las AMPKα1 existe una mayor conservación dentro del género Trypanosoma, mientras que con la AMPKα2 se observó mayor semejanza entre las secuencias de T. cruzi con las Leishmanias. En un análisis más amplio, incluyendo un número mayor de especies pertenecientes al grupo Excavata, determinamos que existe una mayor similitud entre la TcAMPKα2 y las AMPKα de otros organismos de este grupo, incluyendo algunos que presentan una única subunidad α, lo que indicaría que la AMPKα2 es la subunidad canónica. Finalmente, mediante estudios de evolución molecular implementados para detectar eventos de adaptación episódica luego de la divergencia de las isoformas determinamos que ambas proteínas estuvieron sujetas a un fuerte proceso de selección positiva. Estos resultados indicarían que existió una sub-funcionalización de ambas isoformas luego de la divergencia de AMPKα1 y AMPKα2.Fil: Prego, Alejo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Pisciottano, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Martínez, Cecilia Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXXIII Reunión Anual de la Sociedad Argentina de ProtozoologíaCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Protozoologí

An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression

TbVps15 is required for vesicular transport and cytokinesis in Trypanosoma brucei

The class III phosphatidylinositol 3-kinase (PI3K) Vps34 is an important regulator of key cellular functions, including cell growth, survival, intracellular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with the putative serine/threonine protein kinase Vps15, however, its role in signaling has not been deeply evaluated. Here, we have identified the Vps15 orthologue in Trypanosoma brucei, named TbVps15. Knockdown of TbVps15 expression by interference RNA resulted in inhibition of cell growth and blockage of cytokinesis. Scanning electron microcopy revealed a variety of morphological abnormalities, with enlarged parasites and dividing cells that often exhibited a detached flagellum. Transmission electron microscopy analysis of TbVps15 RNAi cells showed an increase in intracellular vacuoles of the endomembrane system and some cells displayed an enlargement of the flagellar pocket, a common feature of cells defective in endocytosis. Moreover, uptake of dextran, transferrin and Concanavalin A was impaired. Finally, TbVps15 downregulation affected the PI3K activity, supporting the hypothesis that TbVps15 and TbVps34 form a complex as occurs in other organisms. In summary, we propose that TbVps15 has a role in the maintenance of cytokinesis, endocytosis and intracellular trafficking in T. brucei.Fil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Miranda, Kildare. Universidade Federal do Rio de Janeiro; BrasilFil: Sternlieb, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Barrera, Nadia Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Girard Dias, Wendell. Universidade Federal do Rio de Janeiro; BrasilFil: de Souza, Wanderley. Universidade Federal do Rio de Janeiro; Brasil. Plataforma de Microscopia Eletrônica Rudolf Barth IOC; BrasilFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin