An investigation of TgDrpC and its interactions with TgFis1 in Toxoplasma gondii

Abstract

Toxoplasma gondii, the causative agent of toxoplasmosis, affects up to one third of the entire human population. Characterized by rapid asexual division, the early stages of T. gondii infection are dependent on the proper partitioning of organelles among daughter cells. Although critical for parasite replication, little is known about the mechanism of mitochondrial division in T. gondii. Previous studies have demonstrated the requirement of T. gondii dynamin-related protein C (TgDrpC) in mitochondrial fission. Localization of TgDrpC along the periphery of mitochondria and at mitochondrial constriction sites seems to suggest a direct role in fission, yet, both the membrane remodeling activity of TgDrpC and its recruitment to the outer mitochondrial membrane remain unclear. Protein BLAST alignments have confirmed the conservation of human Fis1, a mitochondrial adaptor and recruiter of dynamin-related proteins, in T. gondii. This protein, classified as TgFis1, appears to show close structural overlap with human Fis1, yet the interaction between TgFis1 and TgDrpC has not been reported. This thesis investigates the membrane remodeling activity of TgDrpC and its interaction with TgFis1. I define an expression and purification system for TgDrpC and TgFis1 in BL21 E. coli and perform a membrane lipid screen to assess TgDrpC lipid preference. I carry out subsequent TgDrpC-liposome incubation experiments to examine TgDrpC membrane remodeling activity and co-incubate TgDrpC with TgFis1 to study protein-protein interaction. High binding intensity of TgDrpC to cardiolipin suggests a pronounced role of TgDrpC in mitochondrial dynamics while observation of lipid tubulation and lipid fragments implicate a direct role of TgDrpC in mitochondrial fission. Absence of TgDrpC-TgFis1 complexation implies a lack of binding activity, yet I propose future experiments to further test this interaction. To conclude, I shift to the global scale and discuss my findings in the context of future drug development against toxoplasmosis and apicomplexan parasitic diseases more broadly.