Defining the Activity and Interactions of the Dynamin-Related Protein TgDrpC in Toxoplasma gondii Mitochondrial Fission

Abstract

Toxoplasma gondii, the causative agent of toxoplasmosis, is a globally prevalent apicomplexan parasite capable of causing severe disease, particularly in immunocompromised individuals. Mitochondrial fission is essential for parasite replication and host cell infection, yet the molecular machinery governing this process in T. gondii remains poorly defined. Dynamin-Related Protein C (TgDrpC) has been strongly implicated as the primary driver of mitochondrial division, but its full mechanism of action is unknown. Notably, canonical mitochondrial fission protein adaptors or specific membrane lipids identified to play a role in recruiting or coordinating membrane fission in other eukaryotes are either not reported or not essential in T. gondii, suggesting a potentially parasite-specific mitochondrial fission machinery. This thesis aims to elucidate how TgDrpC interacts with lipid membranes and other proteins to execute mitochondrial fission. A membrane lipid screen revealed membrane lipid preferences of TgDrpC. Protein interaction partners were firstly probed by co-immunoprecipitation using recombinant TgDrpC against mitochondrial and whole-cell lysates. To examine interactions in the endogenous context, whole-cell lysate from host-isolated T. gondii was analyzed using thermal proteome co-aggregation coupled with mass spectrometry, and resulting datasets were integrated with Tapioca-based computational network inference to generate a list of candidate protein interactors. Our investigation of endogenous protein interactions yielded a high-confidence set of uncharacterized hypothetical proteins, many of which lack clear homologs outside apicomplexans, as candidate binding partners. Sequence and structural homology analyses of these candidates further suggest roles in membrane association and protein complex assembly, consistent with functions in mitochondrial remodeling. Together, these results advance our understanding of TgDrpC function in the essential process of mitochondrial fission in T. gondii and also contribute a novel set of potential targets for therapeutic intervention against toxoplasmosis.