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Differentiation of the protozoan parasite  Toxoplasma gondii  into its latent bradyzoite stage is a key event in the parasite’s life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the Tg BRADIN/GRA24  gene leads to enhanced differentiation of the parasite, and the Tg BRADIN/GRA24  knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in  T .  gondii . A better understanding of the proteins and mechanisms regulating  T .  gondii  differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

Yeast Three-Hybrid Screen Identifies TgBRADIN/GRA24 as a Negative Regulator of Toxoplasma gondii Bradyzoite Differentiation