Development of Luciferase Expressing Leishmania donovani Axenic Amastigotes as Primary Model for In Vitro Screening of Antileishmanial Compounds

Abstract

The development of new therapeutic leads against leishmaniasis relies primarily on screening of a large number of compounds on multiplication of clinically irrelevant transgenic promastigotes. The advent of the successful in vitro culture of axenic amastigotes allows the development of transgenic axenic amastigotes as a primary screen which can test compounds in a high throughput mode like promastigotes, still representative of the clinically relevant mammalian amastigotes stage. The present study reports the development of luciferase-tagged axenic amastigotes of Leishmania donovani, the causative agent of Indian Kala-azar, for in vitro drug screening. Luciferase expressing promastigotes were transformed to axenic amastigotes at a low pH and high temperature without the loss of luciferase expression. As compared to transgenic promastigotes, the luciferase expressing axenic amastigotes exhibited more sensitivity to antileishmanial drugs, particularly to pentavalent antimony (~2.8-fold) and also to the test compounds. Hence, the developed luciferase expressing axenic amastigotes make an ideal choice for high throughput drug screening for antileishmanial compounds.

Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

“…Axenic amastigotes have also been screened by means of HTS platforms [9], [51], [52]. However, expression arrays comparing both axenic amastigotes and those isolated from infected macrophages have shown metabolic differences, impaired intracellular transport and altered response to oxidative stress [53].…”

High-Content Assay for Measuring Intracellular Growth ofLeishmaniain Human Macrophages

“…[15][16][17] These screens generally employ transgenic Leishmania lines expressing either fluorescent (e.g., GFP) or bioluminescent (e.g., luciferase) reporter proteins and quantify intracellular growth in infected macrophages from total well fluorescence/luminescence, or the shift in host cell fluorescence by flow cytometry. 15,[18][19][20][21][22][23][24][25] However, per-well measurements cannot distinguish between intracellular and extracellular parasites, and in some studies, the reported IC 50 s for known drugs, using a flow cytometry-based method, are considerably greater than those reported with other assays. 21 More recent studies have used high-content screening (HCS) approaches to monitor the intracellular growth of transgenic parasite lines within individual macrophages, providing greater insights into host-parasite interactions and drug toxicity.…”

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant <i>Leishmania major</i> Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

“…Several studies about intracellular pathogenic microorganisms like Leishmania have indicated that independent reporter genes, such as GFP or luciferase, practically facilitate antimicrobial drug and cell biological activity research both in vitro and in vivo [ 7 , 8 , 20 , 25 , 26 ]. Dual reporter gene-expressing pathogens were then generated to more benefit the reporter activity [ 21 , 22 , 27 ].…”

“…GFP is intrinsically fluorescent and can be detected easily and directly, without the need for any extra processing, by several instruments such as the fluorescent microscope, fluorimeter, and fluorescence-activated cell sorting (FACS). Among different reporter genes, LUC has been introduced as a more efficient reporter in the HTS mode [ 16 , 19 ] for drug screening [ 20 ] due to its high sensitivity and low background.…”