In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase

dc.creatorAlonso, Victoria Lucía
dc.creatorManarin, Romina
dc.creatorPerdomo, Virginia Gabriela
dc.creatorGulin, Julián Ernesto Nicolás
dc.creatorSerra, Esteban C.
dc.creatorCribb, Pamela
dc.date2022-12-20T16:52:59Z
dc.date2022-12-20T16:52:59Z
dc.date2021-11
dc.date2022-12-20T16:52:59Z
dc.date2022-12-20T16:52:59Z
dc.date2021-11
dc.date.accessioned2023-08-30T21:17:36Z
dc.date.available2023-08-30T21:17:36Z
dc.identifier1940-087X
dc.identifierhttp://hdl.handle.net/2133/25068
dc.identifierhttp://hdl.handle.net/2133/25068
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8541951
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).
dc.descriptionTrypanosoma 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.descriptionFil: Alonso, Victoria Lucia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.descriptionFil: Manarin, Romina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.descriptionFil: Perdomo, Virginia Gabriela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.descriptionFil: Serra, Esteban C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.descriptionFil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.
dc.descriptionFil: Serra, Esteban. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.descriptionFil: Cribb, Pamela. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.descriptionFil: Gulin, Julián Ernesto Nicolás. Universidad de Buenos Aires. Instituto de Investigaciones Biomédicas (INBIOMED); Argentina.
dc.formatapplication/pdf
dc.languageeng
dc.publisherMyJove Corporation
dc.relationhttps://europepmc.org/article/med/34806703
dc.relationhttps://doi.org/10.3791/63210
dc.rightshttps://creativecommons.org/licenses/by-nc/4.0/deed.es
dc.rightsAlonso, Victoria Lucia
dc.rightsManarin, Romina
dc.rightsPerdomo, Virginia Gabriela
dc.rightsGulin, Julián Ernesto Nicolás
dc.rightsSerra, Esteban C.
dc.rightsCribb, Pamela
dc.rightsAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)
dc.rightsembargoedAccess
dc.subjectChlorocebus aethiops
dc.subjectLife cycle stages
dc.subjectbeta-Galactosidase
dc.subjectDrug evaluation
dc.subjectMammals
dc.subjectVero cells
dc.subjectParasites
dc.titleIn Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase
dc.typearticle
dc.typeartículo


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