title: ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst.
abstract: Cellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.
authors.affiliation: Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Radke
authors.forename: Olivier
authors.initials: O
authors.lastname: Lucas
authors.forename: Erandi K
authors.initials: EK
authors.lastname: De Silva
authors.forename: YanFen
authors.initials: Y
authors.lastname: Ma
authors.forename: William J
authors.initials: WJ
authors.lastname: Sullivan
authors.forename: Louis M
authors.initials: LM
authors.lastname: Weiss
authors.forename: Manuel
authors.initials: M
authors.lastname: Llinas
authors.forename: Michael W
authors.initials: MW
authors.lastname: White
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003560:Cysts / Q000469:parasitology*; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D055786:Gene Knockout Techniques; D006801:Humans; D008156:Luciferases; D052940:Merozoites / Q000254:growth & development* / Q000378:metabolism; D046228:Microarray Analysis; D015800:Protozoan Proteins / Q000378:metabolism*; D014122:Toxoplasma / Q000254:growth & development* / Q000378:metabolism; D014123:Toxoplasmosis / Q000503:physiopathology*; D050656:Transcription Factor AP-2 / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D015800:Protozoan Proteins; D050656:Transcription Factor AP-2; D008156:Luciferases