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

Autor: Gulin JEN; Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Gobierno de la Ciudad de Buenos Aires (GCBA), Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Servicio de Parasitología y Enfermedad de Chagas, Hospital de Niños 'Dr. Ricardo Gutiérrez', Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Instituto de Investigaciones Biomédicas (INBIOMED), Universidad de Buenos Aires (UBA) Facultad de Medicina-CONICET, Paraguay 2155 (C1121ABG), Buenos Aires, Argentina., Rocco DM; Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Gobierno de la Ciudad de Buenos Aires (GCBA), Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Servicio de Parasitología y Enfermedad de Chagas, Hospital de Niños 'Dr. Ricardo Gutiérrez', Gallo 1330 (C1425EFD), Buenos Aires, Argentina., Alonso V; Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (IBR-CONICET-UNR), Suipacha 531 (2000), Rosario, Argentina., Cribb P; Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (IBR-CONICET-UNR), Suipacha 531 (2000), Rosario, Argentina., Altcheh J; Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Gobierno de la Ciudad de Buenos Aires (GCBA), Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Servicio de Parasitología y Enfermedad de Chagas, Hospital de Niños 'Dr. Ricardo Gutiérrez', Gallo 1330 (C1425EFD), Buenos Aires, Argentina., García-Bournissen F; Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Gobierno de la Ciudad de Buenos Aires (GCBA), Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Servicio de Parasitología y Enfermedad de Chagas, Hospital de Niños 'Dr. Ricardo Gutiérrez', Gallo 1330 (C1425EFD), Buenos Aires, Argentina.; Division of Paediatric Clinical Pharmacology, Department of Paediatrics, Schulich School of Medicine & Dentistry, University of Western Ontario, 800 Commissioners Rd. E., Rm. B1-437, London, Canada.
Jazyk: angličtina
Zdroj: Biology methods & protocols [Biol Methods Protoc] 2021 Jul 13; Vol. 6 (1), pp. bpab004. Date of Electronic Publication: 2021 Jul 13 (Print Publication: 2021).
DOI: 10.1093/biomethods/bpab004
Abstrakt: There 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 β-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 µM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 µM, 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 β-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition obtained with the colourimetric assay were 2.31 µM for BZ and 0.97 µM for NFX, similar to the reported values for the Dm28c wild strain (2.80 and 1.5 µM, 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.
(© The Author(s) 2021. Published by Oxford University Press.)
Databáze: MEDLINE
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