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Examinando por Autor "Cribb, Pamela"

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    Construction of three new Gateway® expression plasmids for Trypanosoma cruzi
    (Instituto Oswaldo Cruz, 2014-12) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Serra, Esteban Carlos
    We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
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    In vitro drug screening against all life cycle stages of Trypanosoma cruzi using parasites expressing β-galactosidase
    (MyJove Corporation, 2021-11) Alonso, Victoria Lucía; Manarin, Romina; Perdomo, Virginia Gabriela; Gulin, Julián Ernesto Nicolás; Serra, Esteban Carlos; Cribb, Pamela
    Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi. T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission. This paper describes an assay based on measuring the activity of the cytoplasmic β-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red β-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a β-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.
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    Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain
    (Oxford University Press, 2021-07-13) Gulin, Julián Ernesto Nicolás; Rocco, Daniela Marisa; Alonso, Victoria Lucía; Cribb, Pamela; Altcheh, Jaime; García-Bournissen, Facundo
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    Overexpression of bromodomain factor 3 in Trypanosoma cruzi (TcBDF3) affects differentiation of the parasite and protects it against bromodomain inhibitors
    (Wiley, 2016-06-06) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Cricco, Julia Alejandra; Serra, Esteban Carlos; Glaxo Smith Kline (GSK): provide I-BET151
    The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin. In the present work we show that parasites overexpressing TcBDF3 exhibit altered differentiation patterns and are less susceptible to treatment with bromodomain inhibitors. We also demonstrate that recombinant TcBDF3 is able to bind to these inhibitors in vitro in a concentration-dependant manner. In parallel, the overexpression of a mutated version of TcBDF3 negatively affects growth of epimastigotes. Recent results, including the ones presented here, suggest that bromodomain inhibitors can be conceived as a new type of anti-parasitic drug against trypanosomiasis.
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    Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion
    (Public Library of Science (PLOS), 2015-04-15) Ritagliati, Carla; Alonso, Victoria Lucía; Manarin, Romina; Cribb, Pamela; Serra, Esteban Carlos
    Abstract Background Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. Showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. Methodology Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. Conclusion TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.
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    Overexpression of Trypanosoma cruzi high mobility group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    (Springer Nature, 2019-01-17) Tavernelli, Luis Emilio; Motta, María Cristina M.; Silva Gonçalves, Camila; Santos da Silva, Marcelo; Elías, María Carolina; Alonso, Victoria Lucía; Serra, Esteban Carlos; Cribb, Pamela; Dr. De Gaudenzi, Javier: provide the pTcINDEX-eGFP strain
    Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.
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    Quorum and light signals modulate acetoin/butanediol catabolism in Acinetobacter spp
    (Frontiers Media, 2019-06-20) Tuttobene, Marisel Romina; Fernández-García, Laura; Blasco, Lucía; Cribb, Pamela; Ambroa, Anton; Müller, Gabriela Leticia; Fernández-Cuenca, Felipe; Bleriot, Inés; Rodríguez, Ramiro Esteban; Barbosa, Beatriz G. V.; Lopez-Rojas, Rafael; Trastoy, Rocío; López, María; Bou, Germán; Tomás, María; Mussi, María Alejandra
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    Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells
    (Public Library of Science (PLOS), 2017-02-08) Cribb, Pamela; Perdomo, Virginia Gabriela; Alonso, Victoria Lucía; Manarin, Romina; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Tavernelli, Luis Emilio; Hernández-Pando, Rogelio