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 is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

“…In 2001, it was reported that EGFP expression by recombinant Leishmania mexicana delayed lesion development (Bennett et al 2001;Millington et al 2010). In contrast, recombinant Leishmania amazonensis GFP kept the infectivity potential (Rocha et al 2013) and recombinant L. amazonensis LUC showed more infectivity but not significant in comparison with wild-type (Reimão et al 2013). However, neither of these studies evaluated immune response factors correlating with infectivity of these recombinant parasites.…”

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

“…Even though flow cytometry has the disadvantage over fluorometer plate readers of only providing end-point data, it permits measuring different fluorescent signals on the same cell event. In our hands as well as in recent reports, sensitivity of the fluorometer requires large amounts of fluorescent parasites or high infection rates that do not mimic physiological situations as well as prolonged culture times (Rocha et al, 2013). The assay described here uses non-irradiated dividing cells are used and can be adapted to any cell type (i.e.…”

Cytotoxicity andIn VitroAntileishmanial Activity of Antimony (V), Bismuth (V), and Tin (IV) Complexes of Lapachol

“…Macrophages were exposed to stationary phase promastigotes (2 × 10 6 /well) at a final ratio of 1 : 10. The plates were incubated at 37°C, 5% CO 2, for 5 h in BOD to allow internalization of parasites [13]. Then, the medium was removed for the remaining noninternalized parasites.…”