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

Tatsuki Sugi, Tatsunori Masatani, Fumi Murakoshi, Shin Ichiro Kawazu, Kentaro Kato

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)9-11
Number of pages3
JournalAnalytical Biochemistry
Volume464
DOIs
Publication statusPublished - 2014 Nov 1
Externally publishedYes

Keywords

  • Bradyzoite
  • DUAL luciferase assay
  • High-throughput screening
  • Reporter parasite
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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