Date of Award

Spring 2021

Project Type

Thesis

Program or Major

Microbiology

Degree Name

Master of Science

First Advisor

Victoria Jeffers

Second Advisor

Sherine Elsawa

Third Advisor

Sarah Walker

Abstract

Toxoplasmosis, caused by the parasite Toxoplasma gondii, is a highly prevalent disease found on every content. An estimated 20-40% of the United States’ population is infected with the parasite according to IgG seroprevalence. There are two stages of Toxoplasma that cause disease, tachyzoites which cause the acute phase and the bradyzoites which cause the chronic phase. The chronic stage will persist for a life-long infection which can reactivate to tachyzoites if the host becomes immunocompromised, leading to encephalitis and heart failure. Current drug therapies clinically approved to treat Toxoplasma can only target tachyzoites and can cause adverse reactions. Therefore, the development of novel drugs to target bradyzoites is critical. Histone acetylation is a key post-translational modification involved in active transcription, cell cycle regulation, and DNA repair. There are three points of inhibition in histone acetylation; lysine acetyltransferases, lysine deacetylase, and bromodomain proteins. Lysine acetyltransferases and lysine deacetylases have been well characterized while the role of bromodomain proteins in gene regulation remain relatively unknown. There are six bromodomain proteins that are only conserved within the apicomplexan phylum making them ideal drug target due as they are less likely to cause toxicity in humans. Toxoplasma gondii bromodomain protein 5 is a hypothetical protein unique to parasites with an unknown function. I hypothesis that TgBDP5 is involved in the gene regulation of a parasite-specific pathway. To address this hypothesis three aims were investigated. The first aim was to determine the impact of TgBDP5 on tachyzoite viability. The second investigated a potential homologue in Plasmodium falciparum. The third was to identify the role of TgBDP5 in gene regulation under normal tachyzoite conditions. Aim one addressed the impact of TgBDP5 on tachyzoite growth. To accomplish this aim, two parasite strains were generated. The first was the knockout strain to identify the phenotype resulting from the absence of bdp5. The second was the complement strain which restored bdp5 and ensured any phenotype observed in the knockout, is solely due to the absence of bdp5. As an obligate, intracellular parasite the viability of Toxoplasma is measured by its ability to invade host cells, replicate, and egress to cause infection. This process was measured by a plaque assay, where parasites were inoculated in human foreskin fibroblasts and grown over 5 days. Total plaque count and average plaque area were measured confirming that TgBDP5 is non-essential for tachyzoite viability. The second aim investigated the complex bdp5 is involved in. A triple HA tag was integrated at the 3’end of TgBDP5, but a stable line could not be generated. Experimental data from the Jeffers lab has indicated that TgBDP5 is likely in a complex with TgBDP1. The Plasmodium falciparum homologue of bdp1 has been extensively studied. Using bioinformatic analysis the proteins involved in the P. falciparum complex was compared to proteins in Toxoplasma. TgBDP5 was aligned with a possible homologue that is also found in the PfBDP1 complex. The third aim investigated genes that are targeted by TgBDP5. To accomplish this a global transcriptomic analysis was performed on the knockout, complement, and parental strain under normal tachyzoite growth. This was used to identify genes that are differently expressed in the absence of TgBDP5.

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