Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay

Anal Biochem. 2014 Jun 30. pii: S0003-2697(14)00277-2. doi: 10.1016/j.ab.2014.06.018. [Epub ahead of print]

 

Sugi T¹, Masatani T², Murakoshi F³, Kawazu SI¹, Kato K⁴.

 

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10² parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite's alpha-tubulin promoter. Renilla luciferase activity was detected with at least 10² parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs "bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.

PMID: 24991689