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

GULIN, JULIÁN ERNESTO NICOLÁS; ROCCO, DANIELA MARISA; ALONSO, VICTORIA LUCIA; CRIBB, PAMELA; ALTCHEH, JAIME; GARCÍA-BOURNISSEN, FACUNDO

Biology Methods and Protocols

Oxford Academic

Año: 2021

There is an urgent need to develop safe and more effective drugs for Chagas disease, as current treatment relies on benznidazole (BZ) and nifurtmox (NFX), two nitroheterocyclic drugs developed more than forty years ago. Recently, a vast number of biotechnologies have been attempting to scale-up drug screening process by automating the assessment of large compounds libraries. Using the Trypanosoma cruzi Dm28c strain transfected with an E. coli β-galactosidase gene, we have developed and validated an easy, quick, and reliable assay suitable for high throughput drug screening of candidate compounds for Chagas disease treatment.The optimal system conditions were established at parasite : host cell relationship (i.e. multiplicity of infection) = 5:1, with 2 h of interaction and a read out at 4 days post-infection (dpi) after 4 h incubation with the β-galactosidase substrate red Chlorophenol-β-D-galactopyranoside (CPRG). Concomitantly, in vitro studies were carried out to determine sensitivity to BZ and NFX from Dm28c/pLacZ strain trypomastigotes, by comparing conventional labor-intensive microscopy counting method with our colorimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (LC50) were 41.36 and 17.99 µM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 µM, respectively for the colorimetric method. On the other hand, the drug concentrations resulting in 50% amastigote development inhibition (IC50) obtained with the colorimetric assay were 2.31 µM for BZ and 0.97 µM for NFX, similar to the results obtained with the Dm28c wild strain.In summary, a colorimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up to improve and optimize drug screening capacity, obtaining biologically meaningful values from BZ and NFX sensitivity. This development can be applied to a panel of several T. cruzi strains to develop a high throughput screening program of large drug libraries aiming to identify compounds with anti-T. cruzi activity.