An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Abstract

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania. Leishmaniasis, one of the most neglected tropical diseases, affects over 2 million people each year. Visceral leishmaniasis (VL), also known as Kala-azar, is caused by the protozoan parasites Leishmania donovani and Leishmania infantum and is fatal if left untreated. Because existing treatments are often ineffective due to parasite resistance and/or toxicity new drugs are urgently needed. Leishmaniasis is transmitted to humans by the bite of an infected sandfly. In the insect vector, parasites exist as flagellated forms—promastigotes, which infect macrophage cells of the human host, where they differentiate to round forms known as amastigotes. Amastigotes and promastigotes are substantially different from a molecular perspective. Drug discovery for leishmaniasis has traditionally been complicated by the unavailability of validated drug targets and of relevant drug assays. Whole cell-based assays have been widely used, as they bypass the need for a validated target. However, they use the insect form of the parasite; indeed, the human form, the intracellular amastigote, is difficult to obtain in the laboratory in quantities compatible with drug screening. We describe here the technical advances that made it possible to adapt the intracellular amastigote form of L. donovani to a drug assay compatible with high-throughput screening.