Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Toxoplasma gondii is an important parasitic pathogen of the phylum Apicomplexa. Parasite invasion of host cells involves a unique gliding motility mechanism that is dependent on polymerization of parasite actin. However, in non-motile parasites, the majority of actin is monomeric and filaments only assemble upon initiation of gliding motility. Actin filament turnover is crucial for motility as shown by the detrimental effects of jasplakinolide, an agent that stabilizes actin filaments. T. gondii actin: TgACTI) is functionally highly divergent from conventional actin and only polymerizes into very short filaments. To understand why T. gondii actin does not form long filaments and uncover what contributes to its unusual polymerization kinetics, TgACTI filaments were examined using molecular modeling and biochemical assays. Phalloidin binding to parasite actins rescued the short filaments demonstrating that although TgACTI naturally forms short, unstable filaments on its own, it is capable of forming longer, conventional filaments when stabilized. Molecular docking was used to identify divergent residues within apicomplexan actin that may impart filament stability and revealed critical substitutions in the hydrophobic plug and phalloidin-binding pocket that are predicted to affect subunit interactions within the filament. Mutations were made within TgACTI to replace residues unique to apicomplexan actins with those from muscle actin resulting in formation of longer actin filaments in vitro. When this substituted, stabilized actin was expressed within the parasite, it had a deleterious impact on gliding motility exhibited by aberrant forms of both circular and helical gliding combined with a decrease in speed. The ability of TgACTI to polymerize is also controlled by a minimal set of canonical actin-binding proteins. T. gondii encodes two formin proteins, TgFRM1 and TgFRM2, and interaction of these proteins with TgACTI results in a dramatic increase in polymerization. However, T. gondii profilin: TgPRF) acts to sequester TgACTI from the growing filament. Finally, examination of concentration dependent polymerization has revealed evidence that TgACTI is utilizing an isodesmic mechanism for polymerization. Collectively, these results demonstrate that T. gondii has evolved multiple mechanisms for controlling the length of actin filaments within the parasite and these adaptations appear to be critical for productive gliding motility.
Skillman, Kristen, "A Molecular Basis for Divergent, Unstable Actin Filaments in Toxoplasma gondii" (2011). All Theses and Dissertations (ETDs). 642.