Date of Award
2024
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Microbiology and Molecular Genetics
First Advisor
Dimitry Krementsov
Abstract
The obligate intracellular parasite Toxoplasma gondii infects up to one third of the world’s population. Prevalence varies widely depending on location and socioeconomic resources. Exposure to T.gondii in immunocompetent hosts results in lifelong infection due to the parasite transitioning to chronic quiescent tissue cysts, most prevalently found within the brain. Most patients suffer mild to no symptoms, however those who become immunocompromised following infection are at risk of a deadly reactivation of tissue cysts within the brain. Due to the location of these cysts and the cyst wall that surrounds the parasites this stage of the parasite lacks any drug treatments. The current drug therapies in use focus on controlling acute stage T.gondii infection while combating the cause of immunocompetence. This absence of drug treatment for chronic Toxoplasmosis stems from a lack of current investigations into this stage of the parasite. Chronic Toxoplasma gondii parasites, or bradyzoites, are difficult to investigate due to their natural location within the brain and the difficulty for current lab models to induce bradyzoite differentiation from the acute stage of infection. Current models require non-physiologically relevant stress, or animal models, both of which have various limitations.
This work strives to understand the chronic stage of infection by using a novel brain cell line to induce bradyzoite differentiation. An RNA sequencing experiment within this cell line identified two genes with unknown function expressed within the bradyzoite stage: Microneme 13 and Rhoptry 28. The current understanding of microneme and rhoptry organelles comes from study of the acute stage of the parasite, or tachyzoite. In tachyzoites, these genes are responsible for the invasion of T.gondii into new host cells, and are important for host pathogen interactions. Bradyzoites exhibit decreased growth and invasion; therefore these genes were interesting to our work. We hypothesized they were important for maintenance of bradyzoites within the host, and that without either of these genes the parasite would lack virulence of the bradyzoite. Following CRISPR knockout of each gene, the mutant strains were characterized both in tissue culture and in vivo with a mouse model. From this data, it is clear that the loss of either MIC13 or ROP28 causes defects in growth of the bradyzoite when excysted, and cyst burden is decreased following mouse infection.
This work puts forth a new model for studying chronic stage T.gondii, and indicates two important genes for bradyzoite maintenance within the host. From this further investigation of the mechanism of this decrease in virulence can be studied of MIC13 and ROP28 genes. Also, this model can serve to support many other questions still open within the field and creates the groundwork for a stronger understanding of chronic T. gondii infection that can support future study of drug therapies.
Language
en
Number of Pages
84 p.
Recommended Citation
Goerner, Amber, "Characterization Of Toxoplasma Gondii Genes Mic13 And Rop28 Utilizing A Novel Brain Cell Model For Chronic Infection" (2024). Graduate College Dissertations and Theses. 1970.
https://scholarworks.uvm.edu/graddis/1970