In vitro drug screening against all life cycle stages of Trypanosoma cruzi using parasites expressing β-galactosidase

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dc.creatorAlonso, Victoria Lucía
dc.creatorManarin, Romina
dc.creatorPerdomo, Virginia Gabriela
dc.creatorGulin, Julián Ernesto Nicolás
dc.creatorSerra, Esteban Carlos
dc.creatorCribb, Pamela
dc.date.accessioned2022-12-20T16:52:59Z
dc.date.available2022-12-20T16:52:59Z
dc.date.issued2021-11
dc.descriptionAlonso, V. L., Manarin, R., Perdomo, V., Gulin, E., Serra, E., Cribb, P. In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase. J. Vis. Exp. (177), e63210, doi:10.3791/63210 (2021).es
dc.description.abstractTrypanosoma 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.
dc.description.filFil: Alonso, Victoria Lucia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.description.filFil: Manarin, Romina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.description.filFil: Perdomo, Virginia Gabriela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.description.filFil: Serra, Esteban Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.description.filFil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.description.filFil: Serra, Esteban. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Cribb, Pamela. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Gulin, Julián Ernesto Nicolás. Universidad de Buenos Aires. Instituto de Investigaciones Biomédicas (INBIOMED); Argentina.
dc.description.sponsorshipAgencia Nacional de Promoción Científica y Tecnológica. Ministerio de Ciencia e Innovación Productiva: PICT2016-0439, PICT2019-0526, PICT2019-4212
dc.description.sponsorshipResearch Council United Kingdom: MR/P027989/1
dc.formatapplication/pdf
dc.identifier.issn1940-087X
dc.identifier.urihttp://hdl.handle.net/2133/25068
dc.language.isoenges
dc.publisherMyJove Corporationes
dc.relation.publisherversionhttps://europepmc.org/article/med/34806703
dc.relation.publisherversionhttps://doi.org/10.3791/63210
dc.rightsembargoedAccesses
dc.rights.holderAlonso, Victoria Luciaes
dc.rights.holderManarin, Rominaes
dc.rights.holderPerdomo, Virginia Gabrielaes
dc.rights.holderGulin, Julián Ernesto Nicoláses
dc.rights.holderSerra, Esteban Carloses
dc.rights.holderCribb, Pamelaes
dc.rights.textAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)es
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/deed.es
dc.subjectChlorocebus aethiopses
dc.subjectLife cycle stageses
dc.subjectbeta-Galactosidasees
dc.subjectDrug evaluationes
dc.subjectMammalses
dc.subjectVero cellses
dc.subjectParasiteses
dc.titleIn vitro drug screening against all life cycle stages of Trypanosoma cruzi using parasites expressing β-galactosidasees
dc.typearticle
dc.typeartículo
dc.type.collectionarticulo
dc.type.versionpublishedVersion

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