An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.

Abstract

Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy islabor intensive and subject to observer variation. In this work, we propose afluorimetric method for drugscreening using redfluorescent parasites. FluorescentLeishmania amazonensiswere developed aftertransfection with integration plasmids containing either red (RFP) or greenfluorescent protein (GFP)genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected toflow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents andpropranolol derivatives previously shown to be active againstTrypanosoma cruzi. Flow cytometry analysisdiscriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. Withmicroscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity andsusceptibility to amphotericin B and propranolol derivatives. Retention offluorescence remained in theintracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFPparasites was only achieved in thefluorimeter. Murine BALB/c macrophages were infected with LaRFPparasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) andtested molecules. Although it was possible to determine IC50values for 4 propranolol derivatives (1, 2b, 3, and4b), all compounds were considered inactive. This study is thefirst to develop afluorimetric drug screeningtest forL. amazonensisRFP. Thefluorimetric test was comparable to microscopy with the advantage of beingfaster and not requiring manual counting.