title: Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay.
abstract: 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 10(2) parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite's α-tubulin promoter. Renilla luciferase activity was detected with at least 10(2) 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.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Sugi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Masatani
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Fumi
authors.initials: F
authors.lastname: Murakoshi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kawazu
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: kkato@obihiro.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
mesh_terms: D000818:Animals; D008156:Luciferases / Q000378:metabolism*; D014122:Toxoplasma / Q000254:growth & development*
chemical_list: D008156:Luciferases
keywords: Bradyzoite; DUAL luciferase assay; High-throughput screening; Reporter parasite; Toxoplasma gondii