Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

5-24-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Laurence Sibley

Abstract

Apicomplexan parasites utilize a unique process of rapid motility termed gliding, which is coupled to their invasion of host cells. Gliding and invasion are dependent on parasite actin filaments, yet parasite actin is mostly non-filamentous. Filaments have been detected only transiently during gliding, suggesting that parasite actin filaments are rapidly assembled and disassembled during gliding motility. Little is known about what regulates the turnover of parasite actin filaments. In higher eukaryotes the Actin Depolymerizing Factor: ADF)/Cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin binding proteins conserved in apicomplexan parasites. To investigate the role of ADF in regulating actin dynamics in apicomplexan parasites, Toxoplasma gondii was used as a model apicomplexan, and the activities of T. gondii ADF: TgADF) were analyzed in vitro and in vivo. We found that TgADF engaged in dual activities. In contrast to most ADF/Cofilin proteins, TgADF was found to be a potent actin monomer sequestering protein that strongly inhibited actin polymerization, suggesting that it likely functions to maintain high G-actin concentrations in the cytoplasm of non-motile parasites. This role was reflected in its molecular structure, in which conserved G-actin binding sites were maintained, while key F-actin binding residues were absent. Despite this, TgADF demonstrated the ability to promote actin filament disassembly via a severing mechanism. Using a conditional knockout system we examined the function of TgADF in the parasite. TgADF was found to be essential for controlling productive gliding motility, and its absence lead to defects in host cell invasion, parasite egress, and parasite dispersal. Detailed analysis of motility revealed that parasites were unable to engage in sustained helical gliding, and moved at markedly reduced speeds. These defects are predicted to arise from the presence of more stable actin filaments in the parasite. Overall both the monomer sequestering and filament severing activities of TgADF are predicted to serve important functions in vivo for maintaining high G-actin concentrations for rapid filament assembly, and disassembling actin filaments for rapid filament turnover, respectively. These studies demonstrated that ADF is essential for regulating actin dynamics in T. gondii.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7445JH3

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