UVM Theses and Dissertations
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Author:
Leung, Jacqueline M.
Dept./Program:
Microbiology and Molecular Genetics
Year:
2014
Degree:
PhD
Abstract:
Toxoplasma gondii is an apicomplexan parasite that uses substrate-dependent gliding motility to not only invade cells of its hosts, but also for egress from the infected host cells, for crossing biological barriers and for dissemination through the host organism during infection. Therefore, the ability of the parasite to move is critical for its virulence. T. gondii has been described to engage in three distinct types of gliding motility on coated two-dimensional (2D) surfaces: twirling, circular and helical gliding. The work presented in this dissertation describes a new method for studying motility, and details the molecular basis for how a small molecule perturbs the parameters that define parasite motility in three dimensions (3D).
We show here that motility in a 3D Matrigel-based environment is strikingly different from what is observed in 2D, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility. To test how a change in a parasite's crescent shape might affect trajectory parameters, we compared the motility of [Delta]TgPhIL1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. [Delta]TgPhIL1 mutants exhibited significantly decreased mean trajectory lengths and mean and maximum velocities compared to the parental and complemented parasite lines. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of [Delta]TgPhIL1 parasites in vivo, and demonstrate the power of the quantitative three-dimensional assay for studying parasite motility.
One useful application of this, assay is to determine how small molecules can affect the class XIV myosin motor complex that drives T. gondii motility and invasion. Two of the key components of this complex are a class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. Treatment of parasites with tachypleginA, a small-molecule inhibitor of T. gondii invasion and motility, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity, but identification of the direct target(s) of tachypleginA and the nature of the compound-induced TgMLC1 modification have proven elusive. We report here on a "click chemistry"--Based labelling approach that demonstrated the analogue RT-4 can bind covalently to recombinantly expressed TgMLC1 (rTgMLC1). Mass spectrometry analysis revealed the compound binds directly to and forms an adduct of 225.118 u on Cys58.
Interestingly, studies with model substrates confirmed that the compound can bind covalently to free sulfhydryls. rTgMLC1 expressing a Cys58Ser mutation was refractory to labelling with the biotine-conjugated RT-4 and no longer underwent a mobility shift in response to compound. Furthermore, allelic replacement parasite lines containing the Cys58Ser mutation showed decreased sensitivity to compound treatment in the 3D motility assay. Collectively, these data suggest that the compound binds directly and covalently to TgMLC1 on Cys58. This work represents an important advance in our understanding of the mechanism by which tachyplegin analogues inhibit T. gondii motility, and provides new insights into how this parasite moves in an extracellular matrix-like environment.
We show here that motility in a 3D Matrigel-based environment is strikingly different from what is observed in 2D, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility. To test how a change in a parasite's crescent shape might affect trajectory parameters, we compared the motility of [Delta]TgPhIL1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. [Delta]TgPhIL1 mutants exhibited significantly decreased mean trajectory lengths and mean and maximum velocities compared to the parental and complemented parasite lines. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of [Delta]TgPhIL1 parasites in vivo, and demonstrate the power of the quantitative three-dimensional assay for studying parasite motility.
One useful application of this, assay is to determine how small molecules can affect the class XIV myosin motor complex that drives T. gondii motility and invasion. Two of the key components of this complex are a class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. Treatment of parasites with tachypleginA, a small-molecule inhibitor of T. gondii invasion and motility, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity, but identification of the direct target(s) of tachypleginA and the nature of the compound-induced TgMLC1 modification have proven elusive. We report here on a "click chemistry"--Based labelling approach that demonstrated the analogue RT-4 can bind covalently to recombinantly expressed TgMLC1 (rTgMLC1). Mass spectrometry analysis revealed the compound binds directly to and forms an adduct of 225.118 u on Cys58.
Interestingly, studies with model substrates confirmed that the compound can bind covalently to free sulfhydryls. rTgMLC1 expressing a Cys58Ser mutation was refractory to labelling with the biotine-conjugated RT-4 and no longer underwent a mobility shift in response to compound. Furthermore, allelic replacement parasite lines containing the Cys58Ser mutation showed decreased sensitivity to compound treatment in the 3D motility assay. Collectively, these data suggest that the compound binds directly and covalently to TgMLC1 on Cys58. This work represents an important advance in our understanding of the mechanism by which tachyplegin analogues inhibit T. gondii motility, and provides new insights into how this parasite moves in an extracellular matrix-like environment.