Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain

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dc.citation.titleBiology Methods and Protocols
dc.citation.volume6
dc.creatorGulin, Julián Ernesto Nicolás
dc.creatorRocco, Daniela Marisa
dc.creatorAlonso, Victoria Lucía
dc.creatorCribb, Pamela
dc.creatorAltcheh, Jaime
dc.creatorGarcía-Bournissen, Facundo
dc.date.accessioned2022-03-25T16:13:16Z
dc.date.available2022-03-25T16:13:16Z
dc.date.issued2021-07-13
dc.descriptionThere is an urgent need to develop safer and more effective drugs for Chagas disease, as the current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli b-galactosidase gene, lacZ, we have adapted and validated an easy, quick and reliable in vitro assay suitable for high-throughput screening for candidate compounds with anti-T. cruzi activity. In vitro studies were conducted to determine trypomastigotes sensitivity to BZ and NFX from Dm28c/pLacZ strain by comparing the conventional labour-intensive microscopy counting method with the colourimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (lytic concentration 50%) were 41.36 and 17.99 mM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 mM, for the colourimetric method, respectively. The optimal conditions for the amastigote development inhibitory assay were established considering the parasite–host relationship (i.e. multiplicity of infection) and interaction time, the time for colourimetric readout and the incubation time with the b-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition obtained with the colourimetric assay were 2.31 mM for BZ and 0.97 mM for NFX, similar to the reported values for the Dm28c wild strain (2.80 and 1.5 mM, respectively). In summary, a colourimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up, obtaining biologically meaningful sensibility values with the reference compounds on both trypomastigotes and amastigotes forms. This development could be applied to high-throughput screening programmes aiming to identify compounds with anti-T. cruzi in vitro activity.es
dc.description.filFil: Gulin, Julián Ernesto Nicolás. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.
dc.description.filFil: Gulin, Julián Ernesto Nicolás. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.
dc.description.filFil: Gulin, Julián Ernesto Nicolás. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas (INBIOMED-CONICET); Argentina.
dc.description.filFil: Rocco, Daniela Marisa. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.
dc.description.filFil: Rocco, Daniela Marisa. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.
dc.description.filFil: Alonso, Victoria Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Altcheh, Jaime. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.
dc.description.filFil: Altcheh, Jaime. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.
dc.description.filFil: García-Bournissen, Facundo. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.
dc.description.filFil: García-Bournissen, Facundo. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.
dc.description.filFil: García-Bournissen, Facundo. University of Western Ontario. Schulich School of Medicine & Dentistry. Department of Paediatrics. Division of Paediatric Clinical Pharmacology; Canada.
dc.formatapplication/pdf
dc.format.extent1-8
dc.identifier.issn2396-8923
dc.identifier.urihttp://hdl.handle.net/2133/23254
dc.language.isoenges
dc.publisherOxford University Presses
dc.relation.publisherversionhttps://doi.org/10.1093/biomethods/bpab004
dc.relation.publisherversionhttps://academic.oup.com/biomethods/article/6/1/bpab004/6321447
dc.rightsopenAccesses
dc.rights.holderGulin, Julián Ernesto Nicoláses
dc.rights.holderRocco, Daniela Marisaes
dc.rights.holderAlonso, Victoria Lucíaes
dc.rights.holderCribb, Pamelaes
dc.rights.holderAltcheh, Jaimees
dc.rights.holderGarcía-Bournissen, Facundoes
dc.rights.textAttribution-NonCommercial 4.0 International (CC BY-NC 4.0)es
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/*
dc.subjectTrypanosoma cruzies
dc.subjectIn vitro drug screeninges
dc.subjectHigh-throughput screeninges
dc.titleOptimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi straines
dc.typepublishedVersion
dc.typearticle
dc.typeartículo
dc.type.collectionarticulo
dc.type.versionpublishedVersion

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