Date of Award


Document Type


Degree Name

Master of Science (MS)


Cell and Molecular Biology


Toxoplasma gondii is a single-celled eukaryotic parasite that belongs to the Apicomplexan phylum. Toxoplasma is an obligate intracellular parasite that is related to other important pathogens, such as, Plasmodium, the causative agent of malaria. Toxoplasma can reproduce both sexually and asexually and asexual reproduction is central to the pathogenesis of this organism. Asexual reproduction consists of two developmental stages, the tachyzoites, which grow rapidly and cause the acute infection and the bradyzoites, which grow slowly and cause the chronic infection. We are interested in identifying the genes involved in the bradyzoite differentiation process in order to better understand the biology of the conversion between tachyzoites and bradyzoites. To date, there are no drugs against the bradyzoite form, therefore, understanding which genes are involved in the conversion could lead to improved drug therapies. In the laboratory, we are able to induce the transition from tachyzoites to bradyzoites by starving the parasites of carbon dioxide (CO2). This method of induction renders them unable to synthesize pyrimidines, which stresses the parasites and causes them to switch to the dormant bradyzoite form. Previous work in the Matrajt laboratory, using a forward genetic screen involving insertional mutagenesis, generated several parasite mutants that are unable to switch from tachyzoites to bradyzoites under CO2 starvation conditions. In the current study, we performed whole genome-wide microarray analysis on 7 bradyzoite differentiation mutants and our data suggest that the mutant parasites are ‘stuck’ in an intracellular state even when they are extracellular. This result suggests a commonality of regulation for switching from state to state. We have also characterized functionally related gene sets that are highly represented in the intracellular state and are able to identify genes that express most differently across all mutant strains, as well as, between mutant strains and wild type. Specifically, DNA replication genes are predominantly expressed during the intracellular state compared to the extracellular tachyzoite or bradyzoite states. Interestingly, mutant extracellular tachyzoites also express these DNA replication genes. Lastly, our data suggest a clear difference between the gene expression profiles of wild type extracellular and intracellular parasites. In addition to the microarray studies, we have characterized the disrupted locus in one of the bradyzoite differentiation mutants, mutant B7. A putative non-coding RNA (ncRNA), designated B41, has been physically disrupted in mutant B7 and its expression is developmentally regulated in wild type but reduced in the mutant. B41 is a large, polyadenylated RNA that is alternatively spliced (1kb and 2.2kb) and has no open reading frame. Two genes adjacent to the insertion site, B41 and EST 13803210 have mis-regulated expression in mutant B7. Overexpression studies of EST13803210 suggest that this EST does not play a key role in bradyzoite differentiation. We also used luciferase reporter assays to test if the putative ncRNA, B41, regulates the expression of EST 13803210 and it does not. Although we have been unable to elucidate the function of B41, our results suggest that this gene is responsible for the mutant phenotype since we were able to complement the mutant using a cosmid complementation system that restored the mutant phenotype.