A High-Throughput Amenable Dual Luciferase System for Measuring Toxoplasma gondii Bradyzoite Viability after Drug Treatment

[Image: see text] It is estimated that more than 2 billion people are chronically infected with the intracellular protozoan parasite Toxoplasma gondii (T. gondii). Despite this, there is currently no vaccine to prevent infection in humans, and there is no recognized curative treatment to clear tissue cysts. A major hurdle for identifying effective drug candidates against chronic-stage cysts has been the low throughput of existing in vitro assays for testing the survival of bradyzoites. We have developed a luciferase-based platform for specifically determining bradyzoite survival within in vitro cysts in a 96-well plate format. We engineered a cystogenic type II T. gondii PruΔku80Δhxgpr strain for stage-specific expression of firefly luciferase in the cytosol of bradyzoites and nanoluciferase for secretion into the lumen of the cyst (DuaLuc strain). Using this DuaLuc strain, we found that the ratio of firefly luciferase to nanoluciferase decreased upon treatment with atovaquone or LHVS, two compounds that are known to compromise bradyzoite viability. The 96-well format allowed us to test several additional compounds and generate dose–response curves for calculation of EC(50) values indicating relative effectiveness of a compound. Accordingly, this DuaLuc system should be suitable for screening libraries of diverse compounds and defining the potency of hits or other compounds with a putative antibradyzoite activity.