Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Michael W. White, Ph.D

Committee Member

Dennis E. Kyle, Ph.D.

Committee Member

Jaya Padmanabhan, Ph.D.

Committee Member

Yu Chen, Ph.D.

Keywords

AP2, Apicomplexa, gene expression, regulation, Toxoplasma

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite of animals and man. The asexual life cycle of Toxoplasma involves three very distinct, but tightly coordinated developmental stages. In nature, the sporozoite (contained within an oocyst) and bradyzoite (contained within a tissue cyst) initiate infection of the intermediate host, followed by rapid differentiation into the actively replicating tachyzoite. When countered by an effective host response, the tachyzoite differentiates back into the latent bradyzoite and this unique ability of Toxoplasma to interconvert between the replicating tachyzoite and the latent bradyzoite within a single host is the cause of life long infection. The transcriptional mechanisms controlling tachyzoite to bradyzoite differentiation and inter-conversion are largely unknown, however, a linkage between the parasite cell cycle and differentiation may underlie these developmental mechanisms.

The recent discovery of a family of DNA binding proteins in Apicomplexa (ApiAP2) that are distantly related to the APETALA-2 (AP2) class of plant transcription factors has uncovered an important set of proteins (ApiAP2 factors) that have critical roles in regulating growth and developmental gene expression. Five Toxoplasma ApiAP2s were studied in this thesis project: AP2IX-9, AP2Ib-1, AP2IV-3 (Chapter 2); AP2IV-4 (Chapter 3); and AP2VI-1 (Chapter 4). A major conclusion of this work highlights a novel paradigm in our understanding of the cellular mechanisms regulating stage conversion in Toxoplasma. The study of AP2IX-9 and AP2IV-4 indicate development of the bradyzoite is governed by transcriptional repressors acting at two independent levels, one late in the cell cycle and a second governing the transition from the tachyzoite to the end-stage bradyzoite tissue cyst. The use of repressors to regulate development provides flexibility for the parasite to immediately respond to changing host conditions and modulate the competing needs of expansion and persistence. In addition, the study of AP2VI-1 demonstrates that Toxoplasma employs ApiAP2s that bind chromosome heterochromatin to establish a state of developmental competency

AP2IX-9 (Chapter 2) has a unique transient expression profile restricted to early bradyzoite differentiation, and absent form both the tachyzoite and terminal tissue cyst. Disruption of the AP2IX-9 locus resulted in increased tissue cyst formation in vitro while conditional overexpression of AP2IX-9 significantly reduced 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 BAG1, B-NTPase and LDH2, common markers for bradyzoite development. Two other ApiAP2s, AP2Ib-1 and AP2IV-3 have similar expression profiles as AP2IX-9 and are candidates for expanding our understanding of this repressor mechanisms regulating development.

A number of mRNAs encoding ApiAP2 proteins are dynamically regulated during the tachyzoite cell cycle that also show unique profiles during bradyzoite development. AP2IV-4 (Chapter 3) and AP2VI-1 (Chapter 4) represent two of several cell cycle AP2s whose expression is associated with specific S-phase and mitotic transitions but illustrate divergent roles in regulating growth and development. The expression of AP2IV-4 is exclusive to the tachyzoite stage of development and peak expression coincides with mitosis of the cell cycle. Interestingly, deletion of AP2IV-4 results in the up-regulation of tissue cyst wall and bradyzoite surface antigens in the tachyzoite. The mis-expression of bradyzoite proteins in the tachyzoite indicate AP2IV-4, much like AP2IX-9, is stage specific transcriptional repressor active only late in the tachyzoite cell cycle, likely promoting continued replication of the tachyzoite stage.

For AP2VI-1 (Chapter 4), an S phase exclusive factor, we have verified S phase expression using an HA fusion protein at the endogenous locus, determined its DNA binding specificity by EMSA, and developed a genetic model of conditional expression based on the small molecule, Shield-1. Attempts to genetically delete this factor were successful only in laboratory adapted strains of Toxoplasma, indicating AP2VI-1 has an essential function in the bradyzoite developmental pathway. Genome-wide binding (chromatin immunoprecipitation and microarray analysis (ChIP-chip)) regions of AP2VI-1 are indistinguishable from the recently published CenH3 regions (centromere marker) and similarly fall within the H3K9me2 and H3K9me3 methylation patterns (heterochromatin markers) that mark the centromere boundaries. AP2VI-1 was also detected in mature bradyzoites from in vitro or animal tissue cysts. This dual expression profile for AP2VI-1 may suggest this factor has a unique role in chromosome maintenance or stability during developmental transitions.

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