By Jessica Kamel

Faculty Mentor: Dr. Swati Agrawal

Abstract

Toxoplasma gondii is an obligate intracellular parasite whose pathogenicity depends on the accurate coordination of its organelles. The parasite can infect most warm-blooded animals, including humans, and is best known for causing toxoplasmosis, a disease that can be particularly dangerous for pregnant women and immunocompromised individuals.
The ability to cause the disease depends on the parasite’s ability to coordinate communication between its organelles. One way organelles coordinate their function is through membrane contact sites (MCSs), which are specialized membrane domains where the membranes of two different organelles are brought into close proximity (typically 10-30 nm) by tethering proteins. These sites play essential roles in lipid exchange, calcium signaling, and organelle dynamics. During infection, T.gondii primarily depends on membrane contact sites for inter-organellar communication between the ER, mitochondrion, and apicoplast during infection. MCS may serve as hubs for Ca2+ exchange during the tachyzoite’s calcium-regulated lytic cycle, although their functions remain poorly understood.
Although MCSs are critical in many eukaryotic cells, their molecular composition in apicomplexan parasites such as T.gondii remains poorly understood.
This study aims to define the structural characteristics and subcellular localization of TGME49_232050. We hypothesize that TGME49_232050 plays an essential role in inter-organelle communication through its involvement in MCSs. To test this hypothesis, conditional knockdown T. gondii parasites were generated in a TIR1-expressing parasite line using the auxin – inducible degron (AID) system, allowing regulated depletion of TGME49_232050. To assess the effect of TGME49_232050 depletion of parasite fitness, a plaque assay was performed using confluent human foreskin fibroblast (HFF) monolayers infected with these conditional knockdown parasites. Plaque assay measured the ability of T. gondii to complete its lytic cycle.
In this assay, confluent HFF monolayers were first prepared and then infected with
an equal number of parasites across the two cell lines. Control (TIR1) and conditional knockdown line (MAID 050) were grown in the presence and absence of auxin. After incubation, the cells were stained with crystal violet to visualize plaque formation.
Multiple trials were conducted, but the results were inconsistent. Plaques were observed in both the TIR and TGME49_232050 parasite lines. Some plates showed clear plaque formation primarily under the – auxin condition, while others showed no plaques at all. The absence of plaques on several plates may indicate that a pipetting error resulted in uneven distribution of the parasites across the plates or inaccurate parasite counting. In future experiments, ensuring accurate counting and improving pipetting techniques will be important for obtaining more reliable results.