title: Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.
abstract: 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 TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/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.
authors: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America.
authors: Anahi V
authors: AV
authors: Odell
authors: School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, St Andrews, Fife, Scotland, United Kingdom.
authors: Fanny
authors: F
authors: Tran
authors: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America.
authors: Jenna E
authors: JE
authors: Foderaro
authors: School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, St Andrews, Fife, Scotland, United Kingdom.
authors: Séverine
authors: S
authors: Poupart
authors: School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, St Andrews, Fife, Scotland, United Kingdom.
authors: Ravi
authors: R
authors: Pathak
authors: School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, St Andrews, Fife, Scotland, United Kingdom.
authors: Nicholas J
authors: NJ
authors: Westwood
authors: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America.
authors: Gary E
authors: GE
authors: Ward
mesh_terms: D024509:3' Flanking Region; D002460:Cell Line; D015723:Gene Library; D017125:Genes, Protozoan; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008018:Life Cycle Stages / Q000187:drug effects; D008727:Methotrexate / Q000737:chemistry / Q000494:pharmacology; D010641:Phenotype; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D020798:Two-Hybrid System Techniques
chemical_list: C513588:4-(7-((dimethylamino)methyl)-2-(4-fluorophenyl)imidazo(1,2-a)pyridin-3-yl)pyrimidin-2-amine; D007093:Imidazoles; D015800:Protozoan Proteins; D011743:Pyrimidines; D008727:Methotrexate