Colorimetric Assay for Screening Compounds Against Leishmania Amastigotes Grown in Macrophages

Abstract

An estimated 12 million persons throughout the world suffer from the protozoan disease leishmaniasis. Current treatments have liabilities including poor activity against some forms of leishmaniasis, toxicity, or the need for parenteral administration. Higher throughput methods to screen chemical compounds are needed to facilitate the search for new antileishmania drugs. In the mammalian host, Leishmania parasites exist as amastigotes that replicate within macrophages. Therefore, an in vitro screening assay using intramacrophage amastigotes most closely represents the natural infection. We have transfected strains of Leishmania major and Leishmania amazonensis with the beta-lactamase gene, which catalyzes a colorimetric reaction with the substrate nitrocephin. The growth of these beta-lactamase-expressing Leishmania within macrophages was quantified in 96-well plates using an optical density plate reader, thus simplifying the methodology for scoring inhibitor assays. This simple and relatively inexpensive colorimetric assay helps improve throughput for screening compounds for antileishmania activity.

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular <em>Leishmania donovani </em>Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

“…Many fluorescent dyes and monoclonal antibodies may be employed for flow cytometric assays 10,11 , but these assays are also limited due to less sensitivity and limitation of time interval of drug-treatment to only one day. There are several reporter gene assays available for quantifying the growth of intracellular amastigotes 12,13,14 . An automated screening may be possible using reporter genes, but these assays also have certain drawbacks.…”

“…The way by which the reporter gene is introduced could also influence the physiological properties of the parasite and have an impact on the screening. If the reporter gene is the part of an episomal plasmid, the relative output of reporter may depend on the copy number of the transfected plasmid (which varies from cell to cell) rather than on the activity of the drug 14 . Some reporter parasites which are transformed parasites do not need selective pressure to maintain the reporter gene; however, there could be biological consequences either by disrupting the genomic architecture or just by the presence of the foreign reporter proteins 15 .…”

Comparison of a High-Throughput High-Content Intracellular Leishmania donovani Assay with an Axenic Amastigote Assay

“…With the advent of new technologies, it has now become possible to increase the throughput of the traditionally very labor-intensive intracellular amastigote assays. Two main methods are in use for the detection of intracellular parasites: plate-reader-based methods that rely on reporter constructs (23)(24)(25) and microscopy-based methods that count parasites directly (8,26,27). With the indirect reporterbased assays, there is a risk of artifacts; compounds may interfere with the reporter protein or with the substrate, and no information is obtained regarding the number of host cells or the distribution of amastigotes in macrophages since these are whole-well readout assays.…”

Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

“…Recent progress in automated image-based screening assays (13,17) and genetic engineering of Leishmania spp. (18)(19)(20)(21) has opened important avenues for the high-throughput search of compounds targeting the intracellular stage. Nonetheless, advanced technology and intense manipulation are required to perform these assays, precluding their broad implementation and routine application to clinical isolates.…”