Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology and Molecular Genetics
First Advisor
Gary E. Ward
Abstract
Toxoplasma gondii infection is endemic in the global population. While most infections are asymptomatic, immunocompromised individuals are at increased risk of developing toxoplasmosis disease. A potential new avenue to treat toxoplasmosis is to target host cell invasion, a key step in the parasite’s life cycle. The exocytosis and delivery of rhoptry proteins into the host cell is essential for T. gondii invasion and virulence. The rhoptry secretory apparatus (RSA) has become increasingly well-defined and shows conservation among apicomplexans. While we now know a great deal about both the RSA structure and the individual rhoptry effector proteins that are released, comparatively little is known about how those proteins are delivered into the host cell. Previous electrophysiology experiments revealed that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. We hypothesize that the conductance transient represents a perforation in the host cell plasma membrane through which rhoptry proteins are delivered into the host cell. To further investigate this event, we have developed a higher throughput assay that utilizes high-speed, multi-wavelength, fluorescence imaging to simultaneously visualize host cell perforation and parasite invasion. Using this assay, we have interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking the rhoptry effector RON2 generate the perforation, suggesting that perforation is upstream of effector translocation and moving junction formation. In contrast, parasites lacking components of the rhoptry exocytosis pathway (e.g., RASP2, Nd9) or proteins that regulate rhoptry secretion (e.g., CLAMP), are largely unable to induce the perforation. These data demonstrate that rhoptry exocytosis is required for formation of the perforation. Our results are consistent with a model in which rhoptry exocytosis results in transient perforation of the host cell membrane, enabling the subsequent delivery of rhoptry effector proteins into the host cell. Future work will focus on determining (1) the nature of the perforating agent and (2) whether the perforation is indeed required for rhoptry protein translocation into the host cell.
Language
en
Number of Pages
139 p.
Recommended Citation
Male, Frances K., "Rhoptry-Associated Perforation Of The Host Cell Plasma Membrane During Toxoplasma Gondii Invasion" (2024). Graduate College Dissertations and Theses. 1952.
https://scholarworks.uvm.edu/graddis/1952