Date of Award

Spring 2022

Project Type

Thesis

Program or Major

Genetics

Degree Name

Master of Science

First Advisor

Victoria Jeffers

Second Advisor

Sarah Walker

Third Advisor

Matthew MacManes

Abstract

Toxoplasma gondii is a single celled eukaryotic parasite that causes the disease toxoplasmosis, which infects approximately thirty to fifty percent of the world’s population. Current treatments for toxoplasmosis are lacking and immunocompromised individuals are at risk of a reactivation of acute infection. Tight control of transcription initiation is critical for maintaining proper infection for the parasite. TATA-binding proteins (TBPs) are critical transcription factors for recognizing and binding to the TATA-box and other motifs in eukaryotes to recruit the transcription preinitiation complex. Toxoplasma contains two TBPs, TgTBP1 and TgTBP2, but TATA-boxes or other conserved motifs have not yet been identified in its promoter regions. Little is known about the role of TBPs in Toxoplasma. I hypothesize that TgTBP1 and TgTBP2 bind to specific sequences within promoters, interact with the TFIID complex, and are essential for parasite viability. To investigate the role of TgTBP1 in Toxoplasma, I replaced the endogenous tgtbp1 promoter with a tetracycline regulatable promoter to knockdown tgtbp1. After successfully testing the knockdown system, parasite phenotyping assays were performed. Intracellular plaque assays show that TgTBP1 knockdown is lethal to parasites. Parasite doubling assays show that loss of TgTBP1 results in a significant delay in replication; however, immunofluorescence assays showed that knockdown does not affect cell morphology or halt cell cycle progression, demonstrating that TgTBP1 loss results in slowed progression through the cell cycle. Parasite invasion assays exhibited a defect in host cell invasion upon TgTBP1 knockdown. As tgtbp1 expression decreases, preliminary data shows that tgtbp2 expression increases, providing evidence that TgTBP2 may be attempting to compensate for loss of TgTBP1. With this information, we begin to further narrow down the role of TgTBP1 in transcription initiation and regulation in Toxoplasma gondii.

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